SH3 DOMAIN MEDIATED VIRUS- HOST CELL INTERACTIONS BY THE NONSTRUCTURAL 1 (NS1) OF INFLUENZA A VIRUS

LEENA YLÖSMÄKI

UNIVERSITY OF HELSINKI FACULTY OF MEDICINE SH3 domain mediated virus-host cell interactions by the SH3 domain mediated virus-host cell interactions by the nonstructural protein 1 (NS1) of influenza A virus nonstructural protein 1 (NS1) of influenza A virus

Department of Virology Department of Virology Faculty of Medicine Faculty of Medicine University of Helsinki University of Helsinki Finland Finland

Leena Ylösmäki Leena Ylösmäki

ACADEMIC DISSERTATION ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Medicine, University of Helsinki, To be presented, with the permission of the Faculty of Medicine, University of Helsinki, for public examination in the Lecture Hall 2, Haartmaninkatu 3, for public examination in the Lecture Hall 2, Haartmaninkatu 3, on June 28th, 2016, at 12 noon. on June 28th, 2016, at 12 noon.

Helsinki 2016 Helsinki 2016 SUPERVISED BY: Kalle Saksela, MD, PhD, Professor SUPERVISED BY: Kalle Saksela, MD, PhD, Professor Department of Virology Department of Virology Faculty of Medicine Faculty of Medicine University of Helsinki University of Helsinki Finland Finland

REVIEWED BY: Tero Ahola, PhD, Docent REVIEWED BY: Tero Ahola, PhD, Docent Department of Food and Department of Food and Environmental Sciences Environmental Sciences Faculty of Agriculture and Faculty of Agriculture and Forestry Forestry University of Helsinki University of Helsinki Finland Finland

Denis Kainov, PhD, Docent Denis Kainov, PhD, Docent Institute for Molecular Medicine Institute for Molecular Medicine Finland (FIMM) Finland (FIMM) University of Helsinki University of Helsinki Finland Finland

OPPONENT: Varpu Marjomäki, PhD, Docent OPPONENT: Varpu Marjomäki, PhD, Docent Department of Biological and Department of Biological and Environmental Science Environmental Science Faculty of Mathematics and Faculty of Mathematics and Science Science University of Jyväskylä University of Jyväskylä Finland Finland

ISBN 978-951-51-2283-4 (paperback) ISBN 978-951-51-2283-4 (paperback) ISBN 978-951-51-2284-1 (PDF) ISBN 978-951-51-2284-1 (PDF) Picaset Oy Picaset Oy http://ethesis.helsinki.fi http://ethesis.helsinki.fi Helsinki 2016 Helsinki 2016

2 2 To my family To my family

3 3 Table of Contents Table of Contents

LIST OF ORIGINAL PUBLICATIONS ...... 7 LIST OF ORIGINAL PUBLICATIONS ...... 7 ABSTRACT ...... 8 ABSTRACT ...... 8 ABBREVIATIONS ...... 9 ABBREVIATIONS ...... 9 1 REVIEW OF LITERATURE ...... 11 1 REVIEW OF LITERATURE ...... 11 1.1 Influenza A virus ...... 11 1.1 Influenza A virus ...... 11 1.1.1 Disease and epidemics ...... 11 1.1.1 Disease and epidemics ...... 11 1.1.2 Structure of the virion ...... 12 1.1.2 Structure of the virion ...... 12 1.2 Influenza A virus life cycle ...... 13 1.2 Influenza A virus life cycle ...... 13 1.2.1 Entry ...... 13 1.2.1 Entry ...... 13 1.2.2 Nuclear import of the viral genome...... 14 1.2.2 Nuclear import of the viral genome...... 14 1.2.3 Transcription and replication of the viral genome ...... 14 1.2.3 Transcription and replication of the viral genome ...... 14 1.2.4 Export of the viral genome from the nucleus ...... 15 1.2.4 Export of the viral genome from the nucleus ...... 15 1.2.5 Virion assembly and budding ...... 16 1.2.5 Virion assembly and budding ...... 16 1.3 Influenza A virus NS1 protein ...... 16 1.3 Influenza A virus NS1 protein ...... 16 1.3.1 Stucture of NS1 ...... 16 1.3.1 Stucture of NS1 ...... 16 1.3.2 Intracellular localization of NS1 ...... 17 1.3.2 Intracellular localization of NS1 ...... 17 1.3.3 Post-translational modifications of NS1 ...... 17 1.3.3 Post-translational modifications of NS1 ...... 17 1.3.4 Inhibition of interferon production by NS1 ...... 18 1.3.4 Inhibition of interferon production by NS1 ...... 18 1.3.4.1 Inhibition of the RIG-I pathway ...... 19 1.3.4.1 Inhibition of the RIG-I pathway ...... 19 1.3.4.2 Inhibition of host gene expression ...... 20 1.3.4.2 Inhibition of host gene expression ...... 20 1.3.4.3 JNK pathway ...... 20 1.3.4.3 JNK pathway ...... 20 1.3.4.4 Inhibition of the activity of antiviral PKR and OAS...... 21 1.3.4.4 Inhibition of the activity of antiviral proteins PKR and OAS...... 21 1.3.5 NS1 in apoptotic pathways ...... 22 1.3.5 NS1 in apoptotic pathways ...... 22 1.3.6 Interactions of NS1 with other host cell factors ...... 22 1.3.6 Interactions of NS1 with other host cell factors ...... 22 1.3.6.1 PDZ domain mediated interactions ...... 22 1.3.6.1 PDZ domain mediated interactions ...... 22 1.3.6.2 SH3 domain mediated interactions ...... 23 1.3.6.2 SH3 domain mediated interactions ...... 23 1.3.7 Regulation of viral RNA and protein synthesis by NS1 ...... 23 1.3.7 Regulation of viral RNA and protein synthesis by NS1 ...... 23 1.4 PI3K/Akt pathway ...... 24 1.4 PI3K/Akt pathway ...... 24 1.4.1 An overview of the PI3K/Akt pathway ...... 24 1.4.1 An overview of the PI3K/Akt pathway ...... 24 1.4.2 Targeting of the PI3K/Akt pathway by viruses ...... 25 1.4.2 Targeting of the PI3K/Akt pathway by viruses ...... 25 1.4.3 Induction of the PI3K/Akt pathway by NS1 protein...... 26 1.4.3 Induction of the PI3K/Akt pathway by NS1 protein...... 26 1.5 SH3 domains ...... 27 1.5 SH3 domains ...... 27 1.5.1 SH3 domain structure ...... 28 1.5.1 SH3 domain structure ...... 28 1.5.2 SH3 domain ligand binding motifs ...... 28 1.5.2 SH3 domain ligand binding motifs ...... 28

4 4 1.5.2.1 Typical ligand binding motifs ...... 28 1.5.2.1 Typical ligand binding motifs ...... 28 1.5.2.2 Atypical ligand binding motifs ...... 29 1.5.2.2 Atypical ligand binding motifs ...... 29 1.5.3 Affinity and specificity of SH3 domains ...... 29 1.5.3 Affinity and specificity of SH3 domains ...... 29 1.5.4 Viral proteins as SH3 domain ligands ...... 30 1.5.4 Viral proteins as SH3 domain ligands ...... 30 1.6 Crk adaptor proteins ...... 31 1.6 Crk adaptor proteins ...... 31 1.6.1 Stucture and binding specificities of Crk proteins ...... 32 1.6.1 Stucture and binding specificities of Crk proteins ...... 32 1.6.2 Regulation of Crk proteins ...... 33 1.6.2 Regulation of Crk proteins ...... 33 1.6.3 Nuclear import and export of Crk proteins ...... 34 1.6.3 Nuclear import and export of Crk proteins ...... 34 1.6.4 Biological function of Crk proteins ...... 34 1.6.4 Biological function of Crk proteins ...... 34 1.6.4.1 Crk in PI3K signaling ...... 35 1.6.4.1 Crk in PI3K signaling ...... 35 1.6.4.2 Crk in apoptotic pathways ...... 36 1.6.4.2 Crk in apoptotic pathways ...... 36 2 AIMS OF THE STUDY ...... 37 2 AIMS OF THE STUDY ...... 37 3 MATERIALS AND METHODS ...... 38 3 MATERIALS AND METHODS ...... 38 3.1 Cell culture and transfections (I-III) ...... 38 3.1 Cell culture and transfections (I-III) ...... 38 3.2 Plasmid constructs ...... 38 3.2 Plasmid constructs ...... 38 3.2.1 Eukaryotic expression vectors (I-III) ...... 38 3.2.1 Eukaryotic expression vectors (I-III) ...... 38 3.2.2 Bacterial expression vectors (I,II) ...... 39 3.2.2 Bacterial expression vectors (I,II) ...... 39 3.3 Viruses and virus infections (I,III) ...... 39 3.3 Viruses and virus infections (I,III) ...... 39 3.4 Antibodies (I-III)...... 40 3.4 Antibodies (I-III)...... 40 3.5 Protein sequence alignment (I)...... 40 3.5 Protein sequence alignment (I)...... 40 3.6 Recombinant protein production, purification and phage screening (I,II) ...... 41 3.6 Recombinant protein production, purification and phage screening (I,II) ...... 41 3.7 Protein precipitation and Western blot (I-III) ...... 41 3.7 Protein precipitation and Western blot (I-III) ...... 41 3.8 Detection of phosphorylated proteins (I-III)...... 41 3.8 Detection of phosphorylated proteins (I-III)...... 41 3.9 Dual-luciferase assays (I) ...... 41 3.9 Dual-luciferase assays (I) ...... 41 3.10 In vitro protein binding assays (I,II) ...... 41 3.10 In vitro protein binding assays (I,II) ...... 41 3.11 Cell fractionation (III) ...... 42 3.11 Cell fractionation (III) ...... 42 3.12 Immunostaining and imaging (III) ...... 42 3.12 Immunostaining and imaging (III) ...... 42 4 RESULTS ...... 43 4 RESULTS ...... 43 4.1 Novel SH3 domain interaction partner for IAV NS1 protein (I-III) ...... 43 4.1 Novel SH3 domain interaction partner for IAV NS1 protein (I-III) ...... 43 4.1.1 Characterization of a novel interaction partner for NS1 protein (I-III) ...... 43 4.1.1 Characterization of a novel interaction partner for NS1 protein (I-III) ...... 43 4.1.2 Functional role for the Crk-NS1 interaction (I,II) ...... 44 4.1.2 Functional role for the Crk-NS1 interaction (I,II) ...... 44 4.1.3 Trimeric complex formation upon Crk-NS1 interaction (II) ...... 45 4.1.3 Trimeric complex formation upon Crk-NS1 interaction (II) ...... 45 4.1.4 Functional consequences of the trimeric complex (II)...... 47 4.1.4 Functional consequences of the trimeric complex (II)...... 47 4.2 Localization of the Crk-NS1 complex (III) ...... 47 4.2 Localization of the Crk-NS1 complex (III) ...... 47 4.2.1 NS1 mediates nuclear translocation of Crk proteins (III) ...... 47 4.2.1 NS1 mediates nuclear translocation of Crk proteins (III) ...... 47

5 5 4.2.2 Functional role of Crk translocation into the nucleus by NS1 (III) ...... 47 4.2.2 Functional role of Crk translocation into the nucleus by NS1 (III) ...... 47 5 DISCUSSION ...... 49 5 DISCUSSION ...... 49 5.1 Crk-NS1 interaction and PI3K activation ...... 49 5.1 Crk-NS1 interaction and PI3K activation ...... 49 5.2 Crk-NS1-PI3K complex formation ...... 50 5.2 Crk-NS1-PI3K complex formation ...... 50 5.3 Nuclear localization of Crk-NS1 complex ...... 51 5.3 Nuclear localization of Crk-NS1 complex ...... 51 5.4 Multiple functions of Crk-NS1 interaction...... 52 5.4 Multiple functions of Crk-NS1 interaction...... 52 5.5 Diversity in the SH3 binding motif of NS1 ...... 53 5.5 Diversity in the SH3 binding motif of NS1 ...... 53 5.6 Significance of the NS1 SH3 binding motif for IAV ...... 53 5.6 Significance of the NS1 SH3 binding motif for IAV ...... 53 6 CONCLUSIONS ...... 55 6 CONCLUSIONS ...... 55 7 ACKNOWLEDGEMENTS ...... 56 7 ACKNOWLEDGEMENTS ...... 56 8 REFERENCES ...... 58 8 REFERENCES ...... 58 ORIGINAL PUBLICATIONS ...... 77 ORIGINAL PUBLICATIONS ...... 77

6 6 LIST OF ORIGINAL PUBLICATIONS LIST OF ORIGINAL PUBLICATIONS

I: Heikkinen LS, Kazlauskas A, Melén K, Wagner R, Ziegler T, Julkunen I, and Saksela K. I: Heikkinen LS, Kazlauskas A, Melén K, Wagner R, Ziegler T, Julkunen I, and Saksela K. Avian and 1918 Spanish influenza A virus NS1 proteins bind to Crk/CrkL SH3 domains Avian and 1918 Spanish influenza A virus NS1 proteins bind to Crk/CrkL SH3 domains to activate host cell signaling. to activate host cell signaling. J Biol Chem. 2008 Feb 29;283(9):5719-27. J Biol Chem. 2008 Feb 29;283(9):5719-27.

II: Ylösmäki L, Schmotz C, Ylösmäki E, and Saksela K. II: Ylösmäki L, Schmotz C, Ylösmäki E, and Saksela K. Reorganization of the host cell Crk(L)-PI3 kinase signaling complex by the influenza A Reorganization of the host cell Crk(L)-PI3 kinase signaling complex by the influenza A virus NS1 protein. virus NS1 protein. Virology. 2015 Jun 19;484:146-152. Virology. 2015 Jun 19;484:146-152.

III: Ylösmäki L, Fagerlund R, Kuisma I, Julkunen I, Saksela K. III: Ylösmäki L, Fagerlund R, Kuisma I, Julkunen I, Saksela K. Nuclear translocation of Crk adaptor proteins by the influenza A virus NS1 protein. Nuclear translocation of Crk adaptor proteins by the influenza A virus NS1 protein. Viruses. 2016 Apr 15;8(4). Viruses. 2016 Apr 15;8(4).

7 7 ABSTRACT ABSTRACT

Src homology 3 (SH3) domains are small modular protein structures that recognize and Src homology 3 (SH3) domains are small modular protein structures that recognize and bind to short proline-rich sequence motifs in their ligand proteins. Viral proteins may bind to short proline-rich sequence motifs in their ligand proteins. Viral proteins may also harbor such binding motifs and thereby serve as SH3 ligands in order to regulate also harbor such binding motifs and thereby serve as SH3 ligands in order to regulate the host cell signaling to support virus growth and replication, and to modulate the host cell signaling to support virus growth and replication, and to modulate virulence. The aim of this study was to examine if influenza A virus (IAV) might also use virulence. The aim of this study was to examine if influenza A virus (IAV) might also use this strategy to take control of its host cells, and to characterize possible SH3 domain- this strategy to take control of its host cells, and to characterize possible SH3 domain- containing host cell binding partners of IAV to establish their role in the cell biology of containing host cell binding partners of IAV to establish their role in the cell biology of IAV infection. IAV infection. IAVs cause seasonal epidemics and occasional pandemics that pose a major threat to IAVs cause seasonal epidemics and occasional pandemics that pose a major threat to human health. The nonstructural protein 1 (NS1) is an important virulence factor of IAV. human health. The nonstructural protein 1 (NS1) is an important virulence factor of IAV. It is a multifunctional protein that suppresses the host interferon response via multiple It is a multifunctional protein that suppresses the host interferon response via multiple mechanisms. Another function of NS1 is to activate phosphatidylinositol-3 kinase (PI3K) mechanisms. Another function of NS1 is to activate phosphatidylinositol-3 kinase (PI3K) signaling in the host cell through direct binding to the p85β regulatory subunit of PI3K. signaling in the host cell through direct binding to the p85β regulatory subunit of PI3K. The NS1-induced activation of PI3K is required for efficient replication of many IAV The NS1-induced activation of PI3K is required for efficient replication of many IAV strains. strains. We found that NS1 proteins from some IAV strains contain an SH3 binding site that We found that NS1 proteins from some IAV strains contain an SH3 binding site that mediates strong and selective binding to the N-terminal SH3 (nSH3) domain of Crk- mediates strong and selective binding to the N-terminal SH3 (nSH3) domain of Crk- family proteins, an important class of adaptor proteins involved in the coordination of family proteins, an important class of adaptor proteins involved in the coordination of cellular . This Crk SH3 binding motif was present in the NS1 of cellular signal transduction. This Crk SH3 binding motif was present in the NS1 of infamous 1918 Spanish Flu pandemic virus as well as in many contemporary avian IAV infamous 1918 Spanish Flu pandemic virus as well as in many contemporary avian IAV strains. In contrast, it is not found in most NS1 proteins of seasonal human IAV strains. strains. In contrast, it is not found in most NS1 proteins of seasonal human IAV strains. We found that the capacity of avian and Spanish Flu NS1 proteins to interact with Crk We found that the capacity of avian and Spanish Flu NS1 proteins to interact with Crk SH3 domains provided them with a greatly enhanced capacity to activate PI3K signaling. SH3 domains provided them with a greatly enhanced capacity to activate PI3K signaling. The molecular mechanism underlying this potentiation was found to be due to a The molecular mechanism underlying this potentiation was found to be due to a reorganization of the natural PI3K-Crk complex by the SH3-binding competent NS1 reorganization of the natural PI3K-Crk complex by the SH3-binding competent NS1 protein. Of note, Crk proteins were found to indirectly (via p85β binding) contribute also protein. Of note, Crk proteins were found to indirectly (via p85β binding) contribute also to PI3K regulation by NS1 proteins of common human IAV strains that lack an SH3 to PI3K regulation by NS1 proteins of common human IAV strains that lack an SH3 binding motif and a capacity for direct Crk recruitment. binding motif and a capacity for direct Crk recruitment. Moreover, we found that the role of the NS1/Crk interaction is not limited to PI3K Moreover, we found that the role of the NS1/Crk interaction is not limited to PI3K regulation. We observed that binding of NS1 to the Crk SH3 domain induced a robust regulation. We observed that binding of NS1 to the Crk SH3 domain induced a robust nuclear accumulation of the predominantly cytoplasmic Crk proteins. This nuclear nuclear accumulation of the predominantly cytoplasmic Crk proteins. This nuclear translocation of Crk proteins was shown to lead to a change in tyrosine phosphorylation translocation of Crk proteins was shown to lead to a change in tyrosine phosphorylation pattern of nuclear proteins. pattern of nuclear proteins. In summary, our studies establish Crk adaptor proteins as important cellular co-factors In summary, our studies establish Crk adaptor proteins as important cellular co-factors exploited by the IAV virulence factor NS1 to manipulate host cell signaling. These results exploited by the IAV virulence factor NS1 to manipulate host cell signaling. These results increase our understanding of the role of NS1 in IAV cell biology, and reveal possible increase our understanding of the role of NS1 in IAV cell biology, and reveal possible new targets for future antiviral drug development aimed against critical host cell new targets for future antiviral drug development aimed against critical host cell interactions rather than highly mutable viral proteins. interactions rather than highly mutable viral proteins.

8 8 ABBREVIATIONS ABBREVIATIONS

ATP adenosine triphosphate ATP adenosine triphosphate AP-1 activator protein 1 AP-1 activator protein 1 BAD Bcl2-antagonist of cell death BAD Bcl2-antagonist of cell death C-terminal carboxyterminal C-terminal carboxyterminal CARD caspase recruiting domain CARD caspase recruiting domain CPSF cleavage and polyadenylation specificity factor CPSF cleavage and polyadenylation specificity factor CPSF30 30 kDa subunit of the CPSF complex CPSF30 30 kDa subunit of the CPSF complex CrkL Crk-like CrkL Crk-like CRM1 chromosomal maintenance 1, exportin 1 CRM1 chromosomal maintenance 1, exportin 1 cRNA complementary RNA cRNA complementary RNA cSH3 C-terminal SH3 domain cSH3 C-terminal SH3 domain DNA-PK DNA-dependent protein kinase DNA-PK DNA-dependent protein kinase dsDNA double-stranded DNA dsDNA double-stranded DNA EBV Ebstein-barr virus EBV Ebstein-barr virus ED effector domain ED effector domain EGFR epidermal growth factor receptor EGFR epidermal growth factor receptor eIF4GI elongation and initiation factor 4GI eIF4GI elongation and initiation factor 4GI ER endoplasmic reticulum ER endoplasmic reticulum FAK focal adhesion kinase FAK focal adhesion kinase FasL Fas-ligand FasL Fas-ligand GST gluthathione S-transferase GST gluthathione S-transferase HA hemagglutin HA hemagglutin HCV hepatitis C virus HCV hepatitis C virus HIV-1 human immunodeficiency virus-1 HIV-1 human immunodeficiency virus-1 Hsp90 heat shock protein 90 Hsp90 heat shock protein 90 IAV influenza A virus IAV influenza A virus IFN interferon IFN interferon IRF3 interferon response factor 3 IRF3 interferon response factor 3 ISC inter SH3 core ISC inter SH3 core ISG interferon stimulated gene ISG interferon stimulated gene iSH2 inter SH2 domain iSH2 inter SH2 domain ISRE interferon stimulated response element ISRE interferon stimulated response element JNK c-jun N-terminal kinase JNK c-jun N-terminal kinase Kd dissociation constant Kd dissociation constant LMP1 latent membrane protein 1 LMP1 latent membrane protein 1 M1 matrix protein M1 matrix protein M2 integral membrane protein, ion channel protein M2 integral membrane protein, ion channel protein MBP maltose binding protein MBP maltose binding protein MDA5 melanoma differentiation-associated gene 5 MDA5 melanoma differentiation-associated gene 5 MEF mouse embryonic fibroblast MEF mouse embryonic fibroblast MLK mixed lineage kinase 3 MLK mixed lineage kinase 3 MOI multiplicity of infection MOI multiplicity of infection mRNA messenger RNA mRNA messenger RNA mTORC2 mammalian target of rapamycin complex 2 mTORC2 mammalian target of rapamycin complex 2 N-terminal aminoterminal N-terminal aminoterminal NA neuraminidase NA neuraminidase NFκB nuclear factor κB NFκB nuclear factor κB NEP nuclear export protein NEP nuclear export protein NES nuclear export signal NES nuclear export signal

9 9 NLS nuclear localization signal NLS nuclear localization signal NMR nuclear magnetic resonance NMR nuclear magnetic resonance NoLS nucleolar localization signal NoLS nucleolar localization signal NP nucleoprotein NP nucleoprotein NPC nuclear pore complex NPC nuclear pore complex NS1 nonstructural protein 1 NS1 nonstructural protein 1 NS2 nonstructural protein 2 NS2 nonstructural protein 2 NS5A nonstructural protein 5A NS5A nonstructural protein 5A nSH3 N-terminal SH3 domain nSH3 N-terminal SH3 domain OAS 2’-5’-oligo A synthetase OAS 2’-5’-oligo A synthetase p110 catalytic subunit of PI3K p110 catalytic subunit of PI3K p85 regulatory subunit of PI3K p85 regulatory subunit of PI3K PA, PA-X IAV polymerase subunits PA, PA-X IAV polymerase subunits PABP1 poly(A)-binding protein 1 PABP1 poly(A)-binding protein 1 PB1, PB1-F2 IAV polymerase subunits PB1, PB1-F2 IAV polymerase subunits PB2 IAV polymerase subunit PB2 IAV polymerase subunit PBM PDZ binding motif PBM PDZ binding motif PDK-1 phosphoinositide-dependent kinase-1 PDK-1 phosphoinositide-dependent kinase-1 PEP proline enriched phosphatase PEP proline enriched phosphatase PH pleckstrin homology PH pleckstrin homology PHLPP PH domain and leucine-rich repeat protein phosphatase PHLPP PH domain and leucine-rich repeat protein phosphatase PI3K phosphatidylinositol-3-kinase PI3K phosphatidylinositol-3-kinase PIP2 phosphatidylinositol (3,4)-bisphosphate PIP2 phosphatidylinositol (3,4)-bisphosphate PIP3 phosphatidylinositol (3,4,5)-trisphosphate PIP3 phosphatidylinositol (3,4,5)-trisphosphate PKB protein kinase B; Akt PKB protein kinase B; Akt PKR protein kinase R PKR protein kinase R PPII polyproline-2 PPII polyproline-2 PP2A protein phosphatase 2A PP2A protein phosphatase 2A pre-mRNA precursor messenger RNA pre-mRNA precursor messenger RNA PTEN phosphatase of tensin homologue deleted on chromosome 10 PTEN phosphatase of tensin homologue deleted on chromosome 10 RBD RNA-binding domain RBD RNA-binding domain RIG-I retinoic acid-inducible gene 1 RIG-I retinoic acid-inducible gene 1 RNA ribonucleic acid RNA ribonucleic acid RNP ribonucleoprotein RNP ribonucleoprotein RTK receptor RTK receptor tyrosine kinase SH2 Src homology 2 SH2 Src homology 2 SH3 Src homology 3 SH3 Src homology 3 SKAP55 Src kinase-associated protein of 55 kDa SKAP55 Src kinase-associated protein of 55 kDa SOS son of sevenless SOS son of sevenless ssRNA single-stranded RNA ssRNA single-stranded RNA SUMO small ubiquitin-like modifier SUMO small ubiquitin-like modifier vtRNA vaultRNA vtRNA vaultRNA vRNA viral RNA vRNA viral RNA vRNP viral ribonucleoprotein vRNP viral ribonucleoprotein WT wild-type WT wild-type

10 10 1 REVIEW OF LITERATURE 1 REVIEW OF LITERATURE

1.1 Influenza A virus 1.1 Influenza A virus Influenza A virus (IAV) belongs to the family Orthomyxoviridae, which is a family of Influenza A virus (IAV) belongs to the family Orthomyxoviridae, which is a family of enveloped viruses with segmented single-stranded RNA genome (Lamb and Krug, 2001). enveloped viruses with segmented single-stranded RNA genome (Lamb and Krug, 2001). At the present, the family includes five other virus genera: Influenzavirus B, At the present, the family includes five other virus genera: Influenzavirus B, Influenzavirus C, Thogotovirus, Quaranjavirus, and Isavirus (ICTV, 2014). IAVs are further Influenzavirus C, Thogotovirus, Quaranjavirus, and Isavirus (ICTV, 2014). IAVs are further divided into different subtypes based on the antigenic variation of the surface divided into different subtypes based on the antigenic variation of the surface glycoproteins, the hemagglutin (HA) and the neuraminidase (NA). Currently, there are glycoproteins, the hemagglutin (HA) and the neuraminidase (NA). Currently, there are 18 known subtybes for HA (H1-18), and 11 for NA (N1-11) (Fouchier et al., 2005; Lamb 18 known subtybes for HA (H1-18), and 11 for NA (N1-11) (Fouchier et al., 2005; Lamb and Krug, 2001; Tong et al., 2012; Tong et al., 2013). The HA subtypes 1-16 and NA and Krug, 2001; Tong et al., 2012; Tong et al., 2013). The HA subtypes 1-16 and NA subtypes 1-9 have been isolated from the aquatic wild birds (CDC, 2015). In contrast, subtypes 1-9 have been isolated from the aquatic wild birds (CDC, 2015). In contrast, only a limited number of IAV subtypes have been isolated from humans. In addition to only a limited number of IAV subtypes have been isolated from humans. In addition to aquatic birds and humans, IAVs also infect a variety of animal species, including other aquatic birds and humans, IAVs also infect a variety of animal species, including other birds, pigs, horses, seals, cats, and dogs (Wright and Webster, 2001). Recently, two new birds, pigs, horses, seals, cats, and dogs (Wright and Webster, 2001). Recently, two new subtypes, H17N10, and H18N11 have been isolated form bats (Tong et al., 2012; Tong subtypes, H17N10, and H18N11 have been isolated form bats (Tong et al., 2012; Tong et al., 2013). et al., 2013). The aquatic birds of the orders Anseriformes (ducks, geese, swan, etc.) and The aquatic birds of the orders Anseriformes (ducks, geese, swan, etc.) and Charadriiformes (gulls, terns, waders, etc.) are the major natural reservoir species for Charadriiformes (gulls, terns, waders, etc.) are the major natural reservoir species for IAVs (Webster et al., 1992). In wild aquatic birds, the IAVs infect predominantly the IAVs (Webster et al., 1992). In wild aquatic birds, the IAVs infect predominantly the epithelial cells of the lower intestinal tract. The transmission from bird to bird occurs via epithelial cells of the lower intestinal tract. The transmission from bird to bird occurs via a fecal-oral route and the virus is maintained mainly as asymptomatic infections in the a fecal-oral route and the virus is maintained mainly as asymptomatic infections in the population. In contrast, domesticated birds of the order Galliformes (turkeys, chickens, population. In contrast, domesticated birds of the order Galliformes (turkeys, chickens, quails, etc.) are susceptible for IAV infection after viral adaptation but they are not a quails, etc.) are susceptible for IAV infection after viral adaptation but they are not a reservoir host for the virus. The first mammalian IAV infection was clinically detected in reservoir host for the virus. The first mammalian IAV infection was clinically detected in swine during the Spanish Flu pandemic in 1918 (Taubenberger et al., 2001). Since that, swine during the Spanish Flu pandemic in 1918 (Taubenberger et al., 2001). Since that, IAVs have been isolated from numerous other mammalian host species as well, including IAVs have been isolated from numerous other mammalian host species as well, including humans, horses, dogs, and marine mammals. Swine are considered as a prime humans, horses, dogs, and marine mammals. Swine are considered as a prime intermediate host for generation of novel IAVs which may have pandemic potential to intermediate host for generation of novel IAVs which may have pandemic potential to humans, since they are susceptible for infections with both avian and human IAV strains humans, since they are susceptible for infections with both avian and human IAV strains (Webster et al., 1992). (Webster et al., 1992). The IAV genome is comprised of eight RNA segments. Depending on the virus strain, the The IAV genome is comprised of eight RNA segments. Depending on the virus strain, the eight segments encode up to 10-13 proteins (see Table 1) (Lamb and Krug, 2001). Two eight segments encode up to 10-13 proteins (see Table 1) (Lamb and Krug, 2001). Two major surface glycoproteins HA and NA, the ion channel M2, and the matrix protein M1 major surface glycoproteins HA and NA, the ion channel M2, and the matrix protein M1 make up the structure of the virion (see below). The NP (nucleoprotein) is bound to viral make up the structure of the virion (see below). The NP (nucleoprotein) is bound to viral RNA (vRNA) to make up the viral ribonucleoproteins (vRNPs) to which the three viral RNA (vRNA) to make up the viral ribonucleoproteins (vRNPs) to which the three viral polymerase subunits (PA, PB1 and PB2) are tightly associated. The nuclear export polymerase subunits (PA, PB1 and PB2) are tightly associated. The nuclear export protein (NEP, formerly known as nonstructural protein 2, NS2) is also present in the protein (NEP, formerly known as nonstructural protein 2, NS2) is also present in the virion, whilst nonstructural protein 1 (NS1), PB1-F2, PA-X, and N40 are expressed in the virion, whilst nonstructural protein 1 (NS1), PB1-F2, PA-X, and N40 are expressed in the infected cells, but absent from the virion. Only the segments encoding NS1/NEP and infected cells, but absent from the virion. Only the segments encoding NS1/NEP and M1/M2 are spliced to produce two proteins. M1/M2 are spliced to produce two proteins.

1.1.1 Disease and epidemics 1.1.1 Disease and epidemics In humans, IAV typically causes an acute infection of the upper respiratory tract. IAV In humans, IAV typically causes an acute infection of the upper respiratory tract. IAV infection is often characterized by the sudden onset of high fever, headache, muscle infection is often characterized by the sudden onset of high fever, headache, muscle pain and severe malaise, together with a cough, sore throat, and running nose (Wright pain and severe malaise, together with a cough, sore throat, and running nose (Wright

11 11 and Webster, 2001). IAV causes seasonal epidemics among humans, which are and Webster, 2001). IAV causes seasonal epidemics among humans, which are estimated to result in about 3-5 million cases of severe illness, and about 250 000 - 500 estimated to result in about 3-5 million cases of severe illness, and about 250 000 - 500 000 deaths every year (WHO, 2014). Occasionally, when a novel subtype of IAV emerges, 000 deaths every year (WHO, 2014). Occasionally, when a novel subtype of IAV emerges, typically from an animal origin, global pandemics occur. In the 20th century, there have typically from an animal origin, global pandemics occur. In the 20th century, there have been four such pandemics. The most severe pandemic known of all time occurred in been four such pandemics. The most severe pandemic known of all time occurred in 1918-1919. The famous “Spanish Flu” has been estimated to have clinically affected 1918-1919. The famous “Spanish Flu” has been estimated to have clinically affected approximately 500 million people, and caused the death of over 40 million people (Wang approximately 500 million people, and caused the death of over 40 million people (Wang and Palese, 2013). Pandemics in 1957 (“Asian Flu”) and 1968 (“Hongkong Flu”) were less and Palese, 2013). Pandemics in 1957 (“Asian Flu”) and 1968 (“Hongkong Flu”) were less severe, yet having significant mortality rates. The most recent pandemic occurred in severe, yet having significant mortality rates. The most recent pandemic occurred in 2009, when an antigenically novel H1N1 virus emerged in swine and started to circulate 2009, when an antigenically novel H1N1 virus emerged in swine and started to circulate in humans (Wang and Palese, 2013). This pandemic virus, called the “Swine Flu”, is still in humans (Wang and Palese, 2013). This pandemic virus, called the “Swine Flu”, is still circulating among humans. circulating among humans.

Table 1. Influenza A virus genome segments, polypeptides and their functions. Adapted from Krug and Table 1. Influenza A virus genome segments, polypeptides and their functions. Adapted from Krug and Fodor, 2013. Fodor, 2013.

Segment Gene Protein Amino acids* Protein mass (kDa)* Protein function Segment Gene Protein Amino acids* Protein mass (kDa)* Protein function 1 PB2 PB2 759 84-87 Polymerase subunit, cap-binding 1 PB2 PB2 759 84-87 Polymerase subunit, cap-binding

2 PB1 PB1 757 87-96 Polymerase subunit, nucleotide addition 2 PB1 PB1 757 87-96 Polymerase subunit, nucleotide addition

N40 718 Unknown N40 718 Unknown

PB1-F2 87 Apoptosis regulator/virulence factor PB1-F2 87 Apoptosis regulator/virulence factor

3 PA PA 716 83-85 Polymerase subunit, endonuclease 3 PA PA 716 83-85 Polymerase subunit, endonuclease

PA-X 252 Modulates host response/degrades PA-X 252 Modulates host response/degrades

host cel polymerase II transcripts host cel polymerase II transcripts

4 HA HA 566 63 Surface glycoprotein, receptor binding 4 HA HA 566 63 Surface glycoprotein, receptor binding

5 NP NP 498 50-60 RNP component, viral replication 5 NP NP 498 50-60 RNP component, viral replication

6 NA NA 454 48-63 Surface glycoprotein 6 NA NA 454 48-63 Surface glycoprotein

7 M M1 252 25-28 Matrix protein 7 M M1 252 25-28 Matrix protein

M2 97 11-15 Ion channel M2 97 11-15 Ion channel

8 NS NS1 230 25-27 Nonstructural protein, multifunctional 8 NS NS1 230 25-27 Nonstructural protein, multifunctional

NS2/NEP 121 12-14 Nuclear export protein NS2/NEP 121 12-14 Nuclear export protein

* approximation; there is variation between the strains * approximation; there is variation between the strains

1.1.2 Structure of the virion 1.1.2 Structure of the virion IAV virions can display different shapes, the most common one being close to spherical IAV virions can display different shapes, the most common one being close to spherical with a diameter of 80-120 nm (Fujiyoshi et al., 1994). The virions possess three subviral with a diameter of 80-120 nm (Fujiyoshi et al., 1994). The virions possess three subviral components: the envelope, the matrix layer, and the RNP core (see Figure 1 for components: the envelope, the matrix layer, and the RNP core (see Figure 1 for schematic representation of the IAV virion). The viral envelope is a lipid bilayer derived schematic representation of the IAV virion). The viral envelope is a lipid bilayer derived from the host cell plasma membrane, containing both cholesterol-enriched lipid rafts from the host cell plasma membrane, containing both cholesterol-enriched lipid rafts and non-raft lipids (Lamb and Krug, 2001). Two transmembrane viral proteins, HA and and non-raft lipids (Lamb and Krug, 2001). Two transmembrane viral proteins, HA and NA, stick out from the lipid bilayer. The third transmembrane protein, the viral integral NA, stick out from the lipid bilayer. The third transmembrane protein, the viral integral membrane protein (M2) is located within the lipid bilayer and serves as an ion channel. membrane protein (M2) is located within the lipid bilayer and serves as an ion channel. The viral matrix protein (M1) lies underneath the lipid bilayer, making up the matrix The viral matrix protein (M1) lies underneath the lipid bilayer, making up the matrix layer and linking the viral envelope to the viral RNP core. M1 interacts with the layer and linking the viral envelope to the viral RNP core. M1 interacts with the

12 12 cytoplasmic tails of HA, NA, and M2 on the outer side and the vRNA and NP on the inner cytoplasmic tails of HA, NA, and M2 on the outer side and the vRNA and NP on the inner side (Nayak et al., 2009; Rossman and Lamb, 2011). The IAV genome is segmented into side (Nayak et al., 2009; Rossman and Lamb, 2011). The IAV genome is segmented into eight negative-sense RNA strands, located inside the matrix shell. Each RNA strand is eight negative-sense RNA strands, located inside the matrix shell. Each RNA strand is packed into ribonucleoprotein (RNP) complexes that consists of nucleoproteins (NP) and packed into ribonucleoprotein (RNP) complexes that consists of nucleoproteins (NP) and viral RNA polymerases (PA, PB1 and PB2). viral RNA polymerases (PA, PB1 and PB2).

Figure 1. Schematic diagram of the structure of influenza A virus particle. Adapted from Lamb and Krug, Figure 1. Schematic diagram of the structure of influenza A virus particle. Adapted from Lamb and Krug, 2001. 2001.

1.2 Influenza A virus life cycle 1.2 Influenza A virus life cycle The IAV life cycle can be divided into five stages: 1) virus entry into the host cell; 2) entry The IAV life cycle can be divided into five stages: 1) virus entry into the host cell; 2) entry of viral RNPs (vRNPs) into the nucleus; 3) transcription and replication of the viral of viral RNPs (vRNPs) into the nucleus; 3) transcription and replication of the viral genome; 4) export of the vRNPs from the nucleus; and 5) assembly and budding at the genome; 4) export of the vRNPs from the nucleus; and 5) assembly and budding at the host cell membrane. In the following, the steps are discussed in more detail (see Figure host cell membrane. In the following, the steps are discussed in more detail (see Figure 2 for schematic representation of IAV life cycle). 2 for schematic representation of IAV life cycle).

1.2.1 Entry 1.2.1 Entry The HA of IAV is a homotrimer that binds to sialic acid containing cell surface receptors The HA of IAV is a homotrimer that binds to sialic acid containing cell surface receptors (Lamb and Krug, 2001). The specificity of the HA molecules in binding cell surface sialic (Lamb and Krug, 2001). The specificity of the HA molecules in binding cell surface sialic acid receptors depends on the linkage between sialic acids and the carbohydrates they acid receptors depends on the linkage between sialic acids and the carbohydrates they are bound. There are two of these linkages α(2,3) and α(2,6). IAVs isolated from humans are bound. There are two of these linkages α(2,3) and α(2,6). IAVs isolated from humans recognize preferentially the α(2,6) linkage, whereas those from birds recognize the recognize preferentially the α(2,6) linkage, whereas those from birds recognize the α(2,3) linkage. Pig trachea contain sialic acids linked to carbohydrates with both linkages α(2,3) linkage. Pig trachea contain sialic acids linked to carbohydrates with both linkages and the swine has therefore been considered as a prime mixing vessel for the generation and the swine has therefore been considered as a prime mixing vessel for the generation of IAV of pandemic potential to humans (Webster et al., 1992). of IAV of pandemic potential to humans (Webster et al., 1992). Upon binding to cell surface receptors, virus particles are endocytosed into early Upon binding to cell surface receptors, virus particles are endocytosed into early endosomes. About two thirds of the endocytosed IAV particles have been found to endosomes. About two thirds of the endocytosed IAV particles have been found to associate with clathrin-coated pits, and one third of the viruses are internalized via associate with clathrin-coated pits, and one third of the viruses are internalized via clathrin-independent pathway (Rust et al., 2004). Following the internalization, IAVs are clathrin-independent pathway (Rust et al., 2004). Following the internalization, IAVs are transported to late endosomes where the acidic environment induces the fusion of the transported to late endosomes where the acidic environment induces the fusion of the

13 13 viral and endosomal membrane, following the dissociation and degradation of M1 from viral and endosomal membrane, following the dissociation and degradation of M1 from RNPs and the release of viral genome. RNPs and the release of viral genome.

1.2.2 Nuclear import of the viral genome 1.2.2 Nuclear import of the viral genome Unlike most other negative sense RNA viruses, the transcription and replication of IAV Unlike most other negative sense RNA viruses, the transcription and replication of IAV genome occurs in the nucleus of the infected cell (Herz et al., 1981, Jackson et al., 1982). genome occurs in the nucleus of the infected cell (Herz et al., 1981, Jackson et al., 1982). Thus, in order for IAV to replicate, the vRNPs must enter the nucleus. Since they are too Thus, in order for IAV to replicate, the vRNPs must enter the nucleus. Since they are too large for passive diffusion through nuclear pore complexes (NPCs), an active nuclear large for passive diffusion through nuclear pore complexes (NPCs), an active nuclear transport mechanism is required. The cytoplasmic vRNPs travel into the nucleus by using transport mechanism is required. The cytoplasmic vRNPs travel into the nucleus by using the importin-α - importin-β1 -dependent nuclear import pathway (Eisfeld et al., 2015). the importin-α - importin-β1 -dependent nuclear import pathway (Eisfeld et al., 2015). The transportation involves the recognition of nuclear localization signals (NLSs). All four The transportation involves the recognition of nuclear localization signals (NLSs). All four vRNP associated proteins are reported to contain an NLS, but the NLS in NP seems to be vRNP associated proteins are reported to contain an NLS, but the NLS in NP seems to be sufficient for vRNP nuclear import (Cros et al., 2005). sufficient for vRNP nuclear import (Cros et al., 2005).

Figure 2. Schematic representation of influenza A virus life cycle. See the text for description. Adapted Figure 2. Schematic representation of influenza A virus life cycle. See the text for description. Adapted from (Shi et al., 2014). from (Shi et al., 2014).

1.2.3 Transcription and replication of the viral genome 1.2.3 Transcription and replication of the viral genome After the nuclear import of vRNPs, they are transcribed to positive-sense RNA to After the nuclear import of vRNPs, they are transcribed to positive-sense RNA to produce viral mRNA. This is known as primary transcription. As the mature cellular RNAs, produce viral mRNA. This is known as primary transcription. As the mature cellular RNAs, the viral mRNAs have poly(A) tails, and 5’ caps. The 5’caps originate from cellular mRNA the viral mRNAs have poly(A) tails, and 5’ caps. The 5’caps originate from cellular mRNA by process called “cap-snatching” (Bouloy et al., 1978; Plotch et al., 1979; Plotch et al., by process called “cap-snatching” (Bouloy et al., 1978; Plotch et al., 1979; Plotch et al., 1981). The viral polymerase PB2, which has endonuclease activity, binds to the 5’ 1981). The viral polymerase PB2, which has endonuclease activity, binds to the 5’ methylated caps of cellular mRNAs and steals them to be used as primer for primary methylated caps of cellular mRNAs and steals them to be used as primer for primary transcription of vRNPs. The mechanism of viral mRNA polyadenylation is different from transcription of vRNPs. The mechanism of viral mRNA polyadenylation is different from cellular mRNA polyadenylation. While cellular mRNAs are polyadenylated through cellular mRNA polyadenylation. While cellular mRNAs are polyadenylated through cleavage at the polyadenylation signal by cleavage and polyadenylation specificity factor cleavage at the polyadenylation signal by cleavage and polyadenylation specificity factor (CPSF) and subsequent addition of a poly(A) tail at the 3’end, the polyadenylation of viral (CPSF) and subsequent addition of a poly(A) tail at the 3’end, the polyadenylation of viral mRNA is a result of the polymerase moving back and forth over a stretch of mRNA is a result of the polymerase moving back and forth over a stretch of approximately 17 uridine residues located in the 5’ end of the viral segment (Eisfeld et approximately 17 uridine residues located in the 5’ end of the viral segment (Eisfeld et al., 2015). Only two of the eight viral mRNAs require splicing; segments 7 and 8 (Lamb al., 2015). Only two of the eight viral mRNAs require splicing; segments 7 and 8 (Lamb

14 14 and Krug, 2001). The cellular splicing machinery is responsible for the splicing of the and Krug, 2001). The cellular splicing machinery is responsible for the splicing of the segments. The viral proteins are then translated in the cytoplasm by host cell ribosomes. segments. The viral proteins are then translated in the cytoplasm by host cell ribosomes. The replication of genomic vRNA happens in two steps: the initial synthesis of cRNA (the The replication of genomic vRNA happens in two steps: the initial synthesis of cRNA (the positive-sense RNA strand) that is complementary to the full-length vRNA; and the positive-sense RNA strand) that is complementary to the full-length vRNA; and the copying of cRNA into new negative-sense vRNAs (Lamb and Krug, 2001). The newly copying of cRNA into new negative-sense vRNAs (Lamb and Krug, 2001). The newly synthesized viral mRNA cannot act as a template for new vRNAs as it has a 5’ cap derived synthesized viral mRNA cannot act as a template for new vRNAs as it has a 5’ cap derived from host and it is truncated relative to the full-length genomic segments. The viral from host and it is truncated relative to the full-length genomic segments. The viral polymerase initiates the cRNA synthesis in a cap-independent manner, and must be able polymerase initiates the cRNA synthesis in a cap-independent manner, and must be able to proceed through the polyadenylation site at the 5' end of vRNA. It is not well known, to proceed through the polyadenylation site at the 5' end of vRNA. It is not well known, how the synthesis from viral mRNA is switched to the synthesis of cRNA. Soluble NP is how the synthesis from viral mRNA is switched to the synthesis of cRNA. Soluble NP is required to prevent termination of the synthesis and polyadenylation at the poly(A) site required to prevent termination of the synthesis and polyadenylation at the poly(A) site (Beaton and Krug, 1984). Some host proteins have also been suggested to have a role in (Beaton and Krug, 1984). Some host proteins have also been suggested to have a role in the process. For example, the direct interaction of viral polymerase with the large the process. For example, the direct interaction of viral polymerase with the large subunit of host cell Pol II enzyme (Engelhardt et al., 2005) has been proposed to improve subunit of host cell Pol II enzyme (Engelhardt et al., 2005) has been proposed to improve the availability of capped RNA primers for viral transcription (Martinez-Alonso et al., the availability of capped RNA primers for viral transcription (Martinez-Alonso et al., 2016). In addition, all three viral polymerase subunits (PA, PB1 and PB2) are required for 2016). In addition, all three viral polymerase subunits (PA, PB1 and PB2) are required for efficient synthesis of cRNA (Nakagawa et al., 1996). Finally, the cRNAs are used as a efficient synthesis of cRNA (Nakagawa et al., 1996). Finally, the cRNAs are used as a template to synthesize the negative-strand vRNAs. Soluble NP and polymerase subunits template to synthesize the negative-strand vRNAs. Soluble NP and polymerase subunits are added to the newly formed vRNAs to form vRNP complexes. are added to the newly formed vRNAs to form vRNP complexes.

1.2.4 Export of the viral genome from the nucleus 1.2.4 Export of the viral genome from the nucleus IAV uses the cellular CRM1-dependent nuclear export pathway to actively mediate the IAV uses the cellular CRM1-dependent nuclear export pathway to actively mediate the transport of the newly synthesized vRNPs from the nucleus to the cytoplasm. There are transport of the newly synthesized vRNPs from the nucleus to the cytoplasm. There are two viral proteins essential for the vRNP nuclear export: M1 and NEP (Eisfeld et al., two viral proteins essential for the vRNP nuclear export: M1 and NEP (Eisfeld et al., 2015). The M1 that is bound to the vRNP interacts with NEP which in turn interacts with 2015). The M1 that is bound to the vRNP interacts with NEP which in turn interacts with the CRM1 via two NES (nuclear export signal) sequences, and contributes to the nuclear the CRM1 via two NES (nuclear export signal) sequences, and contributes to the nuclear exit of vRNPs. Recent data implies that in addition to the interactions of NEP with M1 exit of vRNPs. Recent data implies that in addition to the interactions of NEP with M1 and CRM1, NEP also interacts with vRNP associated PB1 subunit of the polymerase and CRM1, NEP also interacts with vRNP associated PB1 subunit of the polymerase complex (Brunotte et al., 2014) but further work is needed to clarify this process. In complex (Brunotte et al., 2014) but further work is needed to clarify this process. In addition, the NP and some cellular protein kinases have been proposed to be involved addition, the NP and some cellular protein kinases have been proposed to be involved in the nuclear export of vRNPs (Eisfeld et al., 2015). in the nuclear export of vRNPs (Eisfeld et al., 2015). Furthermore, there is growing evidence for apoptosis playing a critical role in the nuclear Furthermore, there is growing evidence for apoptosis playing a critical role in the nuclear export of vRNPs. Wurzer et al. reported that inhibition of caspases resulted in reduced export of vRNPs. Wurzer et al. reported that inhibition of caspases resulted in reduced production of infectious virus progeny due to nuclear retention of vRNP complexes production of infectious virus progeny due to nuclear retention of vRNP complexes (Wurzer et al., 2003). This was specifically seen at a later stage of infection and was (Wurzer et al., 2003). This was specifically seen at a later stage of infection and was shown to be caspase 3-dependent. Recently, a study by Mühlbauer et al. showed that shown to be caspase 3-dependent. Recently, a study by Mühlbauer et al. showed that the induction of caspases during IAV infection increases the diffusion limit of nuclear the induction of caspases during IAV infection increases the diffusion limit of nuclear pores allowing the vRNPs to passively diffuse out of the nucleus (Muhlbauer et al., 2015). pores allowing the vRNPs to passively diffuse out of the nucleus (Muhlbauer et al., 2015). These results suggest a model in which the vRNPs may exit the nucleus by two different These results suggest a model in which the vRNPs may exit the nucleus by two different methods depending on the time point of infection. In the intermediate steps of the virus methods depending on the time point of infection. In the intermediate steps of the virus life cycle, the vRNPs are exported by an active export mechanism which involves CRM1. life cycle, the vRNPs are exported by an active export mechanism which involves CRM1. At a later stage of infection, the caspase activity increases leading to the widening of At a later stage of infection, the caspase activity increases leading to the widening of nuclear pores, which allows the passive diffusion of vRNPs from the nucleus. nuclear pores, which allows the passive diffusion of vRNPs from the nucleus.

15 15 1.2.5 Virion assembly and budding 1.2.5 Virion assembly and budding The newly synthesized viral proteins and vRNPs are transported to the lipid raft domains The newly synthesized viral proteins and vRNPs are transported to the lipid raft domains on the apical side of the cell plasma membrane where the virus assembly and budding on the apical side of the cell plasma membrane where the virus assembly and budding occurs (Lamb and Krug, 2001). The transmembrane viral glycoproteins HA and NA as occurs (Lamb and Krug, 2001). The transmembrane viral glycoproteins HA and NA as well as the ion channel M2 span the plasma membrane. On the cytoplasmic face of the well as the ion channel M2 span the plasma membrane. On the cytoplasmic face of the membrane, M1 interacts with the cytoplasmic tails of HA, NA, and M2, and in turn membrane, M1 interacts with the cytoplasmic tails of HA, NA, and M2, and in turn interacts with the vRNPs. interacts with the vRNPs. Initiation of the virion budding is not well known. It has been suggested that the arrival Initiation of the virion budding is not well known. It has been suggested that the arrival of the vRNPs to the cell surface or their interaction with M1 promotes the budding of the vRNPs to the cell surface or their interaction with M1 promotes the budding (Lamb and Krug, 2001; Rossman and Lamb, 2011). Host cell protein UBR4, an ubiquitin (Lamb and Krug, 2001; Rossman and Lamb, 2011). Host cell protein UBR4, an ubiquitin ligase, has been shown to play an important role in the process (Tripathi et al., 2015). ligase, has been shown to play an important role in the process (Tripathi et al., 2015). Binding of UBR4 with viral M2 ion channel promotes transport of viral proteins to the Binding of UBR4 with viral M2 ion channel promotes transport of viral proteins to the cell surface. The exact mechanism of virus budding is also unknown. NA has an cell surface. The exact mechanism of virus budding is also unknown. NA has an important role in viral budding, by cleaving the sialic acid residues from the important role in viral budding, by cleaving the sialic acid residues from the glycoproteins and from the surface of infected cells. This facilitates the efficient release glycoproteins and from the surface of infected cells. This facilitates the efficient release of viral particles from the plasma membrane, and prevents self-aggregation of progeny of viral particles from the plasma membrane, and prevents self-aggregation of progeny virions. Other viral proteins, like HA, M1, and M2, have also been postulated to have virions. Other viral proteins, like HA, M1, and M2, have also been postulated to have different roles in viral budding but their contribution to the process is not clear. different roles in viral budding but their contribution to the process is not clear.

1.3 Influenza A virus NS1 protein 1.3 Influenza A virus NS1 protein NS1 is relatively small protein and it is the main viral antagonist of the innate immune NS1 is relatively small protein and it is the main viral antagonist of the innate immune response during IAV infection (Ayllon and Garcia-Sastre, 2015; Hale et al., 2008c). response during IAV infection (Ayllon and Garcia-Sastre, 2015; Hale et al., 2008c). Despite its small size, NS1 has an outstanding number of described functions and Despite its small size, NS1 has an outstanding number of described functions and interaction partners in the host cell. As the name “nonstuctural” implies, the NS1 protein interaction partners in the host cell. As the name “nonstuctural” implies, the NS1 protein is not a structural component of the IAV virion, instead it is expressed at very high levels is not a structural component of the IAV virion, instead it is expressed at very high levels in infected cells (Krug and Etkind, 1973). in infected cells (Krug and Etkind, 1973).

1.3.1 Stucture of NS1 1.3.1 Stucture of NS1 The shortest vRNA segment (segment 8) is transcribed into two mRNAs, which encode The shortest vRNA segment (segment 8) is transcribed into two mRNAs, which encode the nonstructural protein 1 (NS1) and nuclear export protein (NEP) (Inglis et al., 1979; the nonstructural protein 1 (NS1) and nuclear export protein (NEP) (Inglis et al., 1979; Lamb and Choppin, 1979; Lamb and Krug, 2001). NS1 and NEP share 9 N-terminal amino Lamb and Choppin, 1979; Lamb and Krug, 2001). NS1 and NEP share 9 N-terminal residues, as the NS1 protein is translated from the unspliced primary mRNA acid residues, as the NS1 protein is translated from the unspliced primary mRNA transcript, whereas NEP is translated from a spliced transcript (Lamb and Lai, 1980). The transcript, whereas NEP is translated from a spliced transcript (Lamb and Lai, 1980). The splicing procedure is regulated, and only ~10-15 % of the segment 8 is spliced to encode splicing procedure is regulated, and only ~10-15 % of the segment 8 is spliced to encode NEP (Robb et al., 2010). NEP (Robb et al., 2010). NS1 is a small, approximately 26 kDa, protein that has a strain specific length of 230-237 NS1 is a small, approximately 26 kDa, protein that has a strain specific length of 230-237 amino acids (see Figure 3 for schematic representation of NS1 protein) (Lamb and Krug, amino acids (see Figure 3 for schematic representation of NS1 protein) (Lamb and Krug, 2001). NS1 consists of two distinct globular domains: an aminoterminal (N-terminal) 2001). NS1 consists of two distinct globular domains: an aminoterminal (N-terminal) RNA-binding domain (RBD, amino acids 1-73), and a carboxyterminal (C-terminal) RNA-binding domain (RBD, amino acids 1-73), and a carboxyterminal (C-terminal) effector domain (ED, amino acids 85-230/7) separated by a short linker region (Ayllon effector domain (ED, amino acids 85-230/7) separated by a short linker region (Ayllon and Garcia-Sastre, 2015). The two domains are well conserved within strains but the and Garcia-Sastre, 2015). The two domains are well conserved within strains but the flexible linker region and disordered C-terminal tail display significant sequence flexible linker region and disordered C-terminal tail display significant sequence variability. The strain specific length of the NS1 depends on amino acid deletions and variability. The strain specific length of the NS1 depends on amino acid deletions and insertions in these regions. For example, the linker region of highly pathogenic avian insertions in these regions. For example, the linker region of highly pathogenic avian H5N1 IAV strains isolated after 2000 carry a deletion of five amino acids in the linker H5N1 IAV strains isolated after 2000 carry a deletion of five amino acids in the linker region and the NS1 of human IAVs isolated between 1950 and 1989 has a seven amino region and the NS1 of human IAVs isolated between 1950 and 1989 has a seven amino

16 16 acid extension in the C-terminal tail (Dundon and Capua, 2009; Melen et al., 2007). Two acid extension in the C-terminal tail (Dundon and Capua, 2009; Melen et al., 2007). Two RBD associates to form homodimers with a six-helix antiparallel bundle (Cheng et al., RBD associates to form homodimers with a six-helix antiparallel bundle (Cheng et al., 2009; Liu et al., 1997). Dimerization is essential for its binding to double-stranded RNA 2009; Liu et al., 1997). Dimerization is essential for its binding to double-stranded RNA (dsRNA) (Chien et al., 1997; Qian et al., 1995; Wang et al., 1999). The arginine residue at (dsRNA) (Chien et al., 1997; Qian et al., 1995; Wang et al., 1999). The arginine residue at position 38 is absolutely required (Wang et al., 1999) but other adjacent amino acids position 38 is absolutely required (Wang et al., 1999) but other adjacent amino acids also participate in dsRNA binding (Cheng et al., 2009). The ED, which is formed by seven also participate in dsRNA binding (Cheng et al., 2009). The ED, which is formed by seven β-strands and three α-helices, also dimerizes (Aramini et al., 2011; Hale et al., 2008a). β-strands and three α-helices, also dimerizes (Aramini et al., 2011; Hale et al., 2008a). The interaction between EDs takes place through the highly conserved tryptophan The interaction between EDs takes place through the highly conserved tryptophan residues at position 187 (Kerry et al., 2011). ED binds to numerous cellular proteins and residues at position 187 (Kerry et al., 2011). ED binds to numerous cellular proteins and mediates many NS1 functions (see Figure 3) (Ayllon and Garcia-Sastre, 2015; Hale et al., mediates many NS1 functions (see Figure 3) (Ayllon and Garcia-Sastre, 2015; Hale et al., 2008c). 2008c).

Figure 3. Schematic representation of the primary structure of NS1 protein, together with its known Figure 3. Schematic representation of the primary structure of NS1 protein, together with its known interaction partners. See the text for description and abbreviations. Adapted from Ayllon and Garcia- interaction partners. See the text for description and abbreviations. Adapted from Ayllon and Garcia- Sastre, 2015. Sastre, 2015.

1.3.2 Intracellular localization of NS1 1.3.2 Intracellular localization of NS1 NS1 protein has distinct roles in the nucleus and the cytoplasm. For example, CPSF30 NS1 protein has distinct roles in the nucleus and the cytoplasm. For example, CPSF30 binding takes place in the nucleus, while regulation of RIG-I pathway happens in binding takes place in the nucleus, while regulation of RIG-I pathway happens in cytoplasm. Thus, the regulation of NS1 localization is important. Soon after virus cytoplasm. Thus, the regulation of NS1 localization is important. Soon after virus infection, newly synthesized NS1 accumulates in the nucleus, but is transported to the infection, newly synthesized NS1 accumulates in the nucleus, but is transported to the cytoplasm at late time points of infection. The localization of NS1 is mediated by one or cytoplasm at late time points of infection. The localization of NS1 is mediated by one or two nuclear localization signals (NLS) as well as by one nuclear export signal (NES) two nuclear localization signals (NLS) as well as by one nuclear export signal (NES) (Forbes et al., 2013; Han et al., 2010; Melen et al., 2007). NLS1 is found between amino (Forbes et al., 2013; Han et al., 2010; Melen et al., 2007). NLS1 is found between amino acids 35 and 41, overlapping with the dsRNA binding sequence in RBD (Greenspan et al., acids 35 and 41, overlapping with the dsRNA binding sequence in RBD (Greenspan et al., 1988; Melen et al., 2007). It is well-conserved among IAV strains, while C-terminally 1988; Melen et al., 2007). It is well-conserved among IAV strains, while C-terminally located NLS2 is virus strain-specific (Greenspan et al., 1988; Melen et al., 2007; Melen located NLS2 is virus strain-specific (Greenspan et al., 1988; Melen et al., 2007; Melen et al., 2012). In addition, a virus strain specific nucleolar localization signal (NoLS) has et al., 2012). In addition, a virus strain specific nucleolar localization signal (NoLS) has been identified. NoLs overlaps with NLS2, and it requires additional basic residues at been identified. NoLs overlaps with NLS2, and it requires additional basic residues at positions 224 and 229 (Melen et al., 2007; Melen et al., 2012). The nuclear export of NS1 positions 224 and 229 (Melen et al., 2007; Melen et al., 2012). The nuclear export of NS1 is regulated by NES, which is located between amino acids 138-147, leucine residues 144 is regulated by NES, which is located between amino acids 138-147, leucine residues 144 and 146 being critical for its function (Li et al., 1998). and 146 being critical for its function (Li et al., 1998).

1.3.3 Post-translational modifications of NS1 1.3.3 Post-translational modifications of NS1 NS1 has been found to be modified in three ways: phosphorylation, ISG15 (interferon- NS1 has been found to be modified in three ways: phosphorylation, ISG15 (interferon- stimulated gene 15) modification, and SUMO (small ubiquitin-like modifier) stimulated gene 15) modification, and SUMO (small ubiquitin-like modifier) modification. These modifications have been reported to affect the function of NS1 and modification. These modifications have been reported to affect the function of NS1 and viral replication. viral replication.

17 17 NS1 is phosphorylated at residues S42, S48, T49, and T215 (Hale et al., 2009; Hsiang et NS1 is phosphorylated at residues S42, S48, T49, and T215 (Hale et al., 2009; Hsiang et al., 2012; Kathum et al., 2015). The substitution of T215 to alanine in A/Udorn NS1 al., 2012; Kathum et al., 2015). The substitution of T215 to alanine in A/Udorn NS1 resulted in attenuation of viral replication (Hale et al., 2009). This was shown later not resulted in attenuation of viral replication (Hale et al., 2009). This was shown later not to be because of phosphorylation of this site, but because of a deleterious structural to be because of phosphorylation of this site, but because of a deleterious structural change of NS1 due to the mutation (Hsiang et al., 2012). Out of the other change of NS1 due to the mutation (Hsiang et al., 2012). Out of the other phosphorylated residues, only the phosphorylation of S42 and T49 was shown to have a phosphorylated residues, only the phosphorylation of S42 and T49 was shown to have a relevant role (Hsiang et al., 2012; Kathum et al., 2015). The substitution of residue 42 in relevant role (Hsiang et al., 2012; Kathum et al., 2015). The substitution of residue 42 in A/Udorn strain affected dsRNA binding and consequently replication, while the A/Udorn strain affected dsRNA binding and consequently replication, while the phosphorylation of T49 was shown to inhibit the association of NS1 with dsRNA, RIG-I phosphorylation of T49 was shown to inhibit the association of NS1 with dsRNA, RIG-I and TRIM25. and TRIM25. NS1 has been shown to be conjugated to two ubiquitin-like proteins ISG15, an interferon NS1 has been shown to be conjugated to two ubiquitin-like proteins ISG15, an interferon stimulated ubiquitin homologue (Tang et al., 2010; Zhao et al., 2010) and SUMO (Pal et stimulated ubiquitin homologue (Tang et al., 2010; Zhao et al., 2010) and SUMO (Pal et al., 2010). Lysine residue 41 (K41) in NS1 RBD was reported to be the main target for al., 2010). Lysine residue 41 (K41) in NS1 RBD was reported to be the main target for ISGylation by ISG15, and this modification resulted in disruption of interaction with ISGylation by ISG15, and this modification resulted in disruption of interaction with importin α, but had no effect on the RNA binding activity, which is also mediated by K41 importin α, but had no effect on the RNA binding activity, which is also mediated by K41 residue (Zhao et al., 2010). Two lysine residues in the C-terminal region of NS1, K219 residue (Zhao et al., 2010). Two lysine residues in the C-terminal region of NS1, K219 and K221, have been identified as the sites for sumoylation (Xu et al., 2011). The and K221, have been identified as the sites for sumoylation (Xu et al., 2011). The sumoylation of these sites seems to enhance the stability of the NS1 protein, and sumoylation of these sites seems to enhance the stability of the NS1 protein, and substitution of K to E in these sites in A/WSN strain resulted in slower replication of the substitution of K to E in these sites in A/WSN strain resulted in slower replication of the virus. virus.

1.3.4 Inhibition of interferon production by NS1 1.3.4 Inhibition of interferon production by NS1 Innate interferon (IFN) response is one of the first barriers a virus faces when infecting Innate interferon (IFN) response is one of the first barriers a virus faces when infecting a host cell. It serves as a potent antiviral mechanism that limits the replication and a host cell. It serves as a potent antiviral mechanism that limits the replication and spread of viruses (Randall and Goodbourn, 2008). Interferons are soluble cytokines and spread of viruses (Randall and Goodbourn, 2008). Interferons are soluble cytokines and the expression of IFN-α, IFN-β, IFN-λ, and IFN-γ is induced upon viral infection. They act the expression of IFN-α, IFN-β, IFN-λ, and IFN-γ is induced upon viral infection. They act in both autocrine and paracrine manner to upregulate interferon stimulated genes in both autocrine and paracrine manner to upregulate interferon stimulated genes (ISGs). ISGs are involved in development of an antiviral state in host cell and in alerting (ISGs). ISGs are involved in development of an antiviral state in host cell and in alerting neighboring cells of the incoming thread. Some ISGs may also be induced directly by a neighboring cells of the incoming thread. Some ISGs may also be induced directly by a viral infection. The innate IFN response also activates the specific adaptive immune viral infection. The innate IFN response also activates the specific adaptive immune response. Pathogens have developed a number of different strategies to overcome, response. Pathogens have developed a number of different strategies to overcome, limit, or hide from the IFN system and establish successful infection. limit, or hide from the IFN system and establish successful infection. One of the most important functions of IAV NS1 protein is its role in the inhibition of IFN One of the most important functions of IAV NS1 protein is its role in the inhibition of IFN production. Studies with recombinant IAVs that do not express NS1 or that express production. Studies with recombinant IAVs that do not express NS1 or that express truncated forms of NS1, have revealed the essential role for NS1 to counteract the host truncated forms of NS1, have revealed the essential role for NS1 to counteract the host IFN response (Egorov et al., 1998; Fernandez-Sesma et al., 2006; Garcia-Sastre et al., IFN response (Egorov et al., 1998; Fernandez-Sesma et al., 2006; Garcia-Sastre et al., 1998). The IAV strain A/PR8/8/34 (A/PR8) with NS1 deletion replicated to titres ~1000- 1998). The IAV strain A/PR8/8/34 (A/PR8) with NS1 deletion replicated to titres ~1000- fold lower than wt A/PR8 virus in IFN-competent cells (Garcia-Sastre et al., 1998). By fold lower than wt A/PR8 virus in IFN-competent cells (Garcia-Sastre et al., 1998). By contrast, in IFN-deficient cells, Vero cells, virus lacking the NS1 replicated almost as contrast, in IFN-deficient cells, Vero cells, virus lacking the NS1 replicated almost as effective as the wt virus. Egorov et al. generated IAVs with truncated forms of NS1 with effective as the wt virus. Egorov et al. generated IAVs with truncated forms of NS1 with varying lengths (Egorov et al., 1998). They reported that while the replication of a virus varying lengths (Egorov et al., 1998). They reported that while the replication of a virus with NS1 coding the first 124 amino acids was only slightly affected, the production of with NS1 coding the first 124 amino acids was only slightly affected, the production of viruses with shorter forms of NS1 were markedly lowered in IFN-competent systems. viruses with shorter forms of NS1 were markedly lowered in IFN-competent systems. Mutations of arginine 38 and lysine 41 greatly impaired the ability of NS1 to block Mutations of arginine 38 and lysine 41 greatly impaired the ability of NS1 to block interferon production (Pichlmair et al., 2006). The length of NS1 did not influence virus interferon production (Pichlmair et al., 2006). The length of NS1 did not influence virus replication in IFN-incompetent cells. In addition, cells infected with IAV lacking the NS1 replication in IFN-incompetent cells. In addition, cells infected with IAV lacking the NS1

18 18 produce more IFN-α and IFN-β than wt virus (Fernandez-Sesma et al., 2006). Together, produce more IFN-α and IFN-β than wt virus (Fernandez-Sesma et al., 2006). Together, these studies indicated, and further studies have proved, that NS1 is an important factor these studies indicated, and further studies have proved, that NS1 is an important factor limiting the host innate IFN response during IAV infection. NS1 has been shown to inhibit limiting the host innate IFN response during IAV infection. NS1 has been shown to inhibit host cell interferon production both pre- and post-transcriptionally and different IAV host cell interferon production both pre- and post-transcriptionally and different IAV strains have evolved diverse strategies to counteract host innate IFN-mediated antiviral strains have evolved diverse strategies to counteract host innate IFN-mediated antiviral response with NS1 protein (Hayman et al., 2007; Kuo et al., 2010). response with NS1 protein (Hayman et al., 2007; Kuo et al., 2010).

1.3.4.1 Inhibition of the RIG-I pathway 1.3.4.1 Inhibition of the RIG-I pathway RIG-I (retinoic acid-inducible gene 1) is a cytoplasmic RNA helicase that detects viral RIG-I (retinoic acid-inducible gene 1) is a cytoplasmic RNA helicase that detects viral genomic single-stranded RNA (ssRNA) bearing 5’ phosphates upon IAV infection, which genomic single-stranded RNA (ssRNA) bearing 5’ phosphates upon IAV infection, which leads to the activation of RIG-I pathway resulting in the induction of the synthesis of leads to the activation of RIG-I pathway resulting in the induction of the synthesis of type I IFNs (Schlee and Hartmann, 2010). The binding of vRNA to RIG-I triggers a type I IFNs (Schlee and Hartmann, 2010). The binding of vRNA to RIG-I triggers a conformational change, exposing two N-terminal caspase recruiting domains (CARDs) conformational change, exposing two N-terminal caspase recruiting domains (CARDs) (Myong et al., 2009). The second CARD of RIG-I is then ubiquitinated by the E3 ligases (Myong et al., 2009). The second CARD of RIG-I is then ubiquitinated by the E3 ligases TRIM25 and RIPLET (Gack et al., 2009; Oshiumi et al., 2009). The activation of RIG-I TRIM25 and RIPLET (Gack et al., 2009; Oshiumi et al., 2009). The activation of RIG-I pathway leads to the phosphorylation and nuclear translocation of transcription factors, pathway leads to the phosphorylation and nuclear translocation of transcription factors, such as IRF3 (interferon response factor 3), AP-1 (activator protein 1) and NFκB (nuclear such as IRF3 (interferon response factor 3), AP-1 (activator protein 1) and NFκB (nuclear factor κB), which drive the transcription of the type I IFN genes (McWhirter et al., 2005). factor κB), which drive the transcription of the type I IFN genes (McWhirter et al., 2005). The NS1 protein of IAV has been shown to inhibit the activation of IRF3 (Talon et al., The NS1 protein of IAV has been shown to inhibit the activation of IRF3 (Talon et al., 2000), NFκB (Wang et al., 2000) and AP-1 (Ludwig et al., 2002). Recently, it has been 2000), NFκB (Wang et al., 2000) and AP-1 (Ludwig et al., 2002). Recently, it has been found that this inhibition takes place at the RIG-I pathway and NS1 has different ways found that this inhibition takes place at the RIG-I pathway and NS1 has different ways to limit the activation to repress the IFN expression. NS1 may inhibit RIG-I signaling by to limit the activation to repress the IFN expression. NS1 may inhibit RIG-I signaling by forming a complex with RIG-I itself (Jureka et al., 2015; Pichlmair et al., 2006) or by forming a complex with RIG-I itself (Jureka et al., 2015; Pichlmair et al., 2006) or by interacting with the ubiquitin ligases TRIM25 (Gack et al., 2009) and RIPLET (Rajsbaum interacting with the ubiquitin ligases TRIM25 (Gack et al., 2009) and RIPLET (Rajsbaum et al., 2012) that are responsible for activation of the RIG-I pathway. Interaction of NS1 et al., 2012) that are responsible for activation of the RIG-I pathway. Interaction of NS1 with TRIM25 blocks the E3 ligase activity on the CARD domains of RIG-I, and mutations with TRIM25 blocks the E3 ligase activity on the CARD domains of RIG-I, and mutations in TRIM25 interaction site in NS1 cause viral attenuation and higher IFN induction (Gack in TRIM25 interaction site in NS1 cause viral attenuation and higher IFN induction (Gack et al., 2009). The association of NS1 with RIPLET also prevents the activation of RIG-I et al., 2009). The association of NS1 with RIPLET also prevents the activation of RIG-I (Rajsbaum et al., 2012). The interaction site responsible for TRIM25 and RIPLET (Rajsbaum et al., 2012). The interaction site responsible for TRIM25 and RIPLET interaction seems to be different, and the interaction properties to these proteins by interaction seems to be different, and the interaction properties to these proteins by NS1 are virus strain specific (Rajsbaum et al., 2012). In addition, the direct interaction NS1 are virus strain specific (Rajsbaum et al., 2012). In addition, the direct interaction between NS1 and RIG-I has so far been reported only for Spanish Flu NS1, whereas the between NS1 and RIG-I has so far been reported only for Spanish Flu NS1, whereas the NS1 from IAV strain A/Udorn/73 (A/Udorn) was unable to interact directly with RIG-I NS1 from IAV strain A/Udorn/73 (A/Udorn) was unable to interact directly with RIG-I (Jureka et al., 2015). Thus, the mechanism of RIG-I inhibition seems to have evolved (Jureka et al., 2015). Thus, the mechanism of RIG-I inhibition seems to have evolved individually in NS1 proteins of different IAV strains. individually in NS1 proteins of different IAV strains. The RNA-binding property of NS1 has also been postulated to be one mechanism for The RNA-binding property of NS1 has also been postulated to be one mechanism for NS1 to limit the activation of RIG-I pathway by sequestering vRNAs away from RIG-I. NS1 to limit the activation of RIG-I pathway by sequestering vRNAs away from RIG-I. However, the affinity of NS1 to RNA is much lower than that of RIG-I (Chien et al., 2004). However, the affinity of NS1 to RNA is much lower than that of RIG-I (Chien et al., 2004). On the other hand, mutations of the important residues for NS1 RNA binding site On the other hand, mutations of the important residues for NS1 RNA binding site (arginine R38 and lysine K41) greatly impair the ability of NS1 to block interferon (arginine R38 and lysine K41) greatly impair the ability of NS1 to block interferon production (Pichlmair et al., 2006). In addition, mutations at these residues also production (Pichlmair et al., 2006). In addition, mutations at these residues also abrogate the interactions of NS1 with RIG-I, TRIM25 and RIPLET (Gack et al., 2009; abrogate the interactions of NS1 with RIG-I, TRIM25 and RIPLET (Gack et al., 2009; Pichlmair et al., 2006; Rajsbaum et al., 2012). Recently, another amino acid in close Pichlmair et al., 2006; Rajsbaum et al., 2012). Recently, another amino acid in close proximity to the RNA-binding site on NS1 has been added to the RIG-I story: threonine proximity to the RNA-binding site on NS1 has been added to the RIG-I story: threonine 49 (T49) (Kathum et al., 2015). This residue was shown to impair the interaction of NS1 49 (T49) (Kathum et al., 2015). This residue was shown to impair the interaction of NS1 with dsRNA, RIG-I and TRIM25 when phosphorylated. The phosphorylation of T49 with dsRNA, RIG-I and TRIM25 when phosphorylated. The phosphorylation of T49

19 19 happens at late time points of infection, and was suggested to act as a spatial-temporal happens at late time points of infection, and was suggested to act as a spatial-temporal regulator of NS1 to direct its function from the inhibition of innate immunity to its other regulator of NS1 to direct its function from the inhibition of innate immunity to its other functions to facilitate IAV infection (Kathum et al., 2015). functions to facilitate IAV infection (Kathum et al., 2015).

1.3.4.2 Inhibition of host gene expression 1.3.4.2 Inhibition of host gene expression The IAV NS1 protein inhibits the production of IFNs and other antiviral proteins also by The IAV NS1 protein inhibits the production of IFNs and other antiviral proteins also by suppressing the host gene expression. The CPSF (Cleavage and Polyadenylation suppressing the host gene expression. The CPSF (Cleavage and Polyadenylation Specificity Factor) catalyzes the addition of poly(A) tails to cellular pre-mRNAs (Colgan Specificity Factor) catalyzes the addition of poly(A) tails to cellular pre-mRNAs (Colgan and Manley, 1997; Wahle and Keller, 1996). NS1 binds to CPSF30, the 30 kDa subunit of and Manley, 1997; Wahle and Keller, 1996). NS1 binds to CPSF30, the 30 kDa subunit of the CPSF complex, and inhibits the 3’ end processing of cellular pre-mRNAs (Nemeroff the CPSF complex, and inhibits the 3’ end processing of cellular pre-mRNAs (Nemeroff et al., 1998). The unprocessed pre-mRNAs accumulate in the nucleus, and cellular mRNA et al., 1998). The unprocessed pre-mRNAs accumulate in the nucleus, and cellular mRNA processing in the cytoplasm is inhibited (Das et al., 2008). Thus, the translation of IFNs processing in the cytoplasm is inhibited (Das et al., 2008). Thus, the translation of IFNs and other antiviral genes is blocked. Since the polyadenylation of viral mRNAs is and other antiviral genes is blocked. Since the polyadenylation of viral mRNAs is catalyzed by the viral polymerase, the viral transcripts are not affected by the repression catalyzed by the viral polymerase, the viral transcripts are not affected by the repression of CPSF (Robertson et al., 1981). The sequestered pre-mRNAs in the nucleus have also of CPSF (Robertson et al., 1981). The sequestered pre-mRNAs in the nucleus have also been suggested to provide a steady pool of capped 5’ ends for cap snatching by the viral been suggested to provide a steady pool of capped 5’ ends for cap snatching by the viral polymerase (Nemeroff et al., 1998). polymerase (Nemeroff et al., 1998). There are several NS1 residues that have been reported to be relevant for the CPSF30 There are several NS1 residues that have been reported to be relevant for the CPSF30 interaction. The interaction is mediated by the conserved hydrophobic residues 184- interaction. The interaction is mediated by the conserved hydrophobic residues 184- 188, of which the W187 residue is also involved in NS1 ED dimerization (Das et al., 2008). 188, of which the W187 residue is also involved in NS1 ED dimerization (Das et al., 2008). Indeed, the dimerization of NS1 is important for the CPSF30 interaction. The interaction Indeed, the dimerization of NS1 is important for the CPSF30 interaction. The interaction is further stabilized by two NS1 amino acids, phenylalanine F103 and methionine M106 is further stabilized by two NS1 amino acids, phenylalanine F103 and methionine M106 (Kainov et al., 2011; Kochs et al., 2007; Twu et al., 2006). Mutation of these two amino (Kainov et al., 2011; Kochs et al., 2007; Twu et al., 2006). Mutation of these two amino acids does not abrogate the interaction, only reduces the binding of NS1 to CPSF30. The acids does not abrogate the interaction, only reduces the binding of NS1 to CPSF30. The capacity to bind CPSF30 and to suppress gene expression varies between different IAV capacity to bind CPSF30 and to suppress gene expression varies between different IAV strains. Most of the avian and all human isolate IAV strains reported from 1998 onward strains. Most of the avian and all human isolate IAV strains reported from 1998 onward share a strong binding capacity to CPSF30 (Ayllon and Garcia-Sastre, 2015). In contrast, share a strong binding capacity to CPSF30 (Ayllon and Garcia-Sastre, 2015). In contrast, the A/PR8 strain that is widely used in laboratories, and the highly pathogenic H5N1 the A/PR8 strain that is widely used in laboratories, and the highly pathogenic H5N1 avian IAV strain involved in the 1997 human outbreak in Hong Kong lack this property avian IAV strain involved in the 1997 human outbreak in Hong Kong lack this property due to amino acid residue substitution at positions 103 and 106 (Kochs et al., 2007; Twu due to amino acid residue substitution at positions 103 and 106 (Kochs et al., 2007; Twu et al., 2006). NS1 of 2009 pandemic (The Swine Flu) IAV is also defective in binding to et al., 2006). NS1 of 2009 pandemic (The Swine Flu) IAV is also defective in binding to CPSF30 due to amino acid substitutions at positions 108, 125, and 189 (Hale et al., CPSF30 due to amino acid substitutions at positions 108, 125, and 189 (Hale et al., 2010c). 2010c). In contrast to CPSF30-inhibition, which un-specifically blocks the expression of all host In contrast to CPSF30-inhibition, which un-specifically blocks the expression of all host genes, some IAV strains have another, specific mechanism for the inhibition of host gene genes, some IAV strains have another, specific mechanism for the inhibition of host gene expression. NS1 proteins of H3N2 IAVs isolated since 1989 have a 7 amino acid extension expression. NS1 proteins of H3N2 IAVs isolated since 1989 have a 7 amino acid extension in their C-terminus (being 237 amino acids long) (Ayllon and Garcia-Sastre, 2015). The in their C-terminus (being 237 amino acids long) (Ayllon and Garcia-Sastre, 2015). The C-terminus of these NS1 proteins contains a sequence, ARSKV, which is very similar to C-terminus of these NS1 proteins contains a sequence, ARSKV, which is very similar to the motif ARTKQ found on histone H3. The tail of NS1 acts as a mimic for H3 and binds the motif ARTKQ found on histone H3. The tail of NS1 acts as a mimic for H3 and binds to PAF1 transcription-elongation complex which regulates genes involved in the IFN to PAF1 transcription-elongation complex which regulates genes involved in the IFN response (Marazzi et al., 2012). Consequently, the elongation of host gene transcription response (Marazzi et al., 2012). Consequently, the elongation of host gene transcription is blocked. is blocked.

1.3.4.3 JNK pathway 1.3.4.3 JNK pathway IAV infection has been reported to trigger the phosphorylation and activation of c-jun IAV infection has been reported to trigger the phosphorylation and activation of c-jun N-terminal kinase (JNK) pathway (Ludwig et al., 2001). JNK phosphorylation leads to N-terminal kinase (JNK) pathway (Ludwig et al., 2001). JNK phosphorylation leads to

20 20 activation of AP-1 transcription factors ATF-2 and c-Jun, which are required for the activation of AP-1 transcription factors ATF-2 and c-Jun, which are required for the induction of IFN-β production. IAV has been reported to activate JNK by two means: induction of IFN-β production. IAV has been reported to activate JNK by two means: directly by NS1 protein or through vRNA recognition by RIG-I (Nacken et al., 2014). The directly by NS1 protein or through vRNA recognition by RIG-I (Nacken et al., 2014). The activation of JNK seems to have a virus-supportive function, since the chemical inhibition activation of JNK seems to have a virus-supportive function, since the chemical inhibition of JNK resulted in decreased virus replication (Nacken et al., 2012). The ability of NS1 to of JNK resulted in decreased virus replication (Nacken et al., 2012). The ability of NS1 to activate JNK depends on subtype specific sequence variation at amino acid 103 (Nacken activate JNK depends on subtype specific sequence variation at amino acid 103 (Nacken et al., 2014). Phenylalanine at this position, the same amino acid involved in stabilization et al., 2014). Phenylalanine at this position, the same amino acid involved in stabilization of CPSF30 interaction, was shown to be important for JNK activation. On the other hand, of CPSF30 interaction, was shown to be important for JNK activation. On the other hand, NS1 protein of some IAV strains have been reported to inhibit JNK activation (see below) NS1 protein of some IAV strains have been reported to inhibit JNK activation (see below) (Hrincius et al., 2010; Ludwig et al., 2002). Partly it may be indirect, due to the ability of (Hrincius et al., 2010; Ludwig et al., 2002). Partly it may be indirect, due to the ability of NS1 to inhibit RIG-I. NS1 to inhibit RIG-I.

1.3.4.4 Inhibition of the activity of antiviral proteins PKR and OAS 1.3.4.4 Inhibition of the activity of antiviral proteins PKR and OAS NS1 limits also interferon response at the post-transcriptional level by inhibiting two ISG NS1 limits also interferon response at the post-transcriptional level by inhibiting two ISG products, PKR (protein kinase R) and OAS (2’-5’-oligo A synthetase). The transcription of products, PKR (protein kinase R) and OAS (2’-5’-oligo A synthetase). The transcription of PKR and OAS is upregulated upon virus infection, they play a key role in host cell antiviral PKR and OAS is upregulated upon virus infection, they play a key role in host cell antiviral response and they are activated by binding to dsRNA (Ayllon and Garcia-Sastre, 2015; response and they are activated by binding to dsRNA (Ayllon and Garcia-Sastre, 2015; Krug, 2015). For PKR, studies with influenza B virus have shown that cytoplasmic vRNP Krug, 2015). For PKR, studies with influenza B virus have shown that cytoplasmic vRNP can function as an activator (Dauber et al., 2009) which is also believed to be true in IAV can function as an activator (Dauber et al., 2009) which is also believed to be true in IAV infected cells (Dauber and Wolff, 2009). The recognition of dsRNA or vRNPs leads to infected cells (Dauber and Wolff, 2009). The recognition of dsRNA or vRNPs leads to autophosphorylation and activation of PKR. The activated PKR then binds and autophosphorylation and activation of PKR. The activated PKR then binds and phosphorylates its best studied target, the eukaryotic translation initiation factor 2 phosphorylates its best studied target, the eukaryotic translation initiation factor 2 (eIF2α), ultimately leading to inhibition of translation initiation. The PKR mediated block (eIF2α), ultimately leading to inhibition of translation initiation. The PKR mediated block of translation impairs viral spread, since the replication of viruses critically depends on of translation impairs viral spread, since the replication of viruses critically depends on the cellular translation machinery. the cellular translation machinery. OAS catalyzes the formation of 2’-5’-poly(A) chains from ATP to activate the otherwise OAS catalyzes the formation of 2’-5’-poly(A) chains from ATP to activate the otherwise latent RNase L, which repress viral infection by degrading single-stranded RNA latent RNase L, which repress viral infection by degrading single-stranded RNA (Silverman, 2007). OAS/RNaseL may also enhance the activation of IFN transcription, (Silverman, 2007). OAS/RNaseL may also enhance the activation of IFN transcription, since the degradation products may bind to and activate RIG-I. Also, the PKR has been since the degradation products may bind to and activate RIG-I. Also, the PKR has been reported to play an additional role in IFN-induction and the host apoptotic responses reported to play an additional role in IFN-induction and the host apoptotic responses (Garcia et al., 2006; Silverman, 2007). (Garcia et al., 2006; Silverman, 2007). For long time, it was believed that NS1 inhibits PKR activation by competing for binding For long time, it was believed that NS1 inhibits PKR activation by competing for binding to dsRNA (Hatada et al., 1999; Lu et al., 1995). However, the activity of PKR is efficiently to dsRNA (Hatada et al., 1999; Lu et al., 1995). However, the activity of PKR is efficiently limited also by RNA-binding defective NS1 (Li et al., 2006; Min and Krug, 2006). In limited also by RNA-binding defective NS1 (Li et al., 2006; Min and Krug, 2006). In addition, the affinity of NS1 to dsRNA is much lower compared to PKR, so the addition, the affinity of NS1 to dsRNA is much lower compared to PKR, so the competition is out ruled (Li et al., 2006). Instead, it has been shown that NS1 prevents competition is out ruled (Li et al., 2006). Instead, it has been shown that NS1 prevents the activation of PKR by direct interaction, and the amino acids 123-127 in NS1 have the activation of PKR by direct interaction, and the amino acids 123-127 in NS1 have been reported to be important (Min et al., 2007). A recent study by Li et al. has provided been reported to be important (Min et al., 2007). A recent study by Li et al. has provided a new insight to the regulation of PKR by NS1 protein (Li et al., 2015). NS1 triggers the a new insight to the regulation of PKR by NS1 protein (Li et al., 2015). NS1 triggers the upregulation of vtRNAs (vaultRNA), which are part of a large ribonucleoprotein particles upregulation of vtRNAs (vaultRNA), which are part of a large ribonucleoprotein particles located in the cytoplasm. This results in inhibition of PKR activity. In contrast, the dsRNA located in the cytoplasm. This results in inhibition of PKR activity. In contrast, the dsRNA binding affinity of OAS is low enough for NS1 to outcompete it (Min and Krug, 2006). binding affinity of OAS is low enough for NS1 to outcompete it (Min and Krug, 2006). Thus, the mechanism for NS1 to inhibit OAS/RNaseL may be by sequestering the dsRNA Thus, the mechanism for NS1 to inhibit OAS/RNaseL may be by sequestering the dsRNA away from OAS. Recently, RNA helicase MDA5 (melanoma differentiation-associated away from OAS. Recently, RNA helicase MDA5 (melanoma differentiation-associated gene 5), a relative of RIG-I, was shown to be involved in the enhancement of the antiviral gene 5), a relative of RIG-I, was shown to be involved in the enhancement of the antiviral

21 21 response through the recognition of the OAS/RNaseL system (Benitez et al., 2015) and response through the recognition of the OAS/RNaseL system (Benitez et al., 2015) and PKR (Pham et al., 2016). PKR (Pham et al., 2016).

1.3.5 NS1 in apoptotic pathways 1.3.5 NS1 in apoptotic pathways Apoptosis is considered to be a cellular antiviral mechanism to limit viral replication. As Apoptosis is considered to be a cellular antiviral mechanism to limit viral replication. As such, IAVs have developed various means by which to regulate this host defense such, IAVs have developed various means by which to regulate this host defense strategy and NS1 is reported to have both pro- and anti-apoptotic functions (Hale et al., strategy and NS1 is reported to have both pro- and anti-apoptotic functions (Hale et al., 2008c). The anti-apoptotic functions of NS1 can be linked to its ability to suppress IFN 2008c). The anti-apoptotic functions of NS1 can be linked to its ability to suppress IFN production (Zhirnov et al., 2002). While A/PR8 IAV with deleted NS1 induced higher production (Zhirnov et al., 2002). While A/PR8 IAV with deleted NS1 induced higher levels of apoptosis than wt A/PR8 virus in IFN-competent system, similar levels of levels of apoptosis than wt A/PR8 virus in IFN-competent system, similar levels of apoptosis was observed in IFN-incompetent system with both viruses. This was apoptosis was observed in IFN-incompetent system with both viruses. This was speculated to indicate that the anti-apoptotic function of NS1 is IFN-dependent. speculated to indicate that the anti-apoptotic function of NS1 is IFN-dependent. As PKR and OAS/RNase L have also been reported to play a role in apoptosis, the As PKR and OAS/RNase L have also been reported to play a role in apoptosis, the inhibition of these proteins by NS1 may lead to suppression of cell death (Min and Krug, inhibition of these proteins by NS1 may lead to suppression of cell death (Min and Krug, 2006; Takizawa et al., 1996). NS1 also inhibits the JNK-pathway, pathway that is linked 2006; Takizawa et al., 1996). NS1 also inhibits the JNK-pathway, pathway that is linked to activation of apoptosis (Hrincius et al., 2010; Ludwig et al., 2002). As well, the to activation of apoptosis (Hrincius et al., 2010; Ludwig et al., 2002). As well, the association of NS1 to Scribble through PDZ-domain binding has been reported to be anti- association of NS1 to Scribble through PDZ-domain binding has been reported to be anti- apoptotic (see below) (Liu et al., 2010). In addition, activation of the host cell PI3-kinase apoptotic (see below) (Liu et al., 2010). In addition, activation of the host cell PI3-kinase (PI3K) pathway (see below) has been described as an additional, IFN-independent (PI3K) pathway (see below) has been described as an additional, IFN-independent method by which NS1 may limit the induction of apoptosis (Ehrhardt et al., 2007; Shin method by which NS1 may limit the induction of apoptosis (Ehrhardt et al., 2007; Shin et al., 2007b; Zhirnov and Klenk, 2007). However, NS1 has also been reported to have et al., 2007b; Zhirnov and Klenk, 2007). However, NS1 has also been reported to have pro-apoptotic functions. Transient expression of NS1 from several human, swine and pro-apoptotic functions. Transient expression of NS1 from several human, swine and avian IAV isolates have been reported to induce apoptosis in cultured cells (Han et al., avian IAV isolates have been reported to induce apoptosis in cultured cells (Han et al., 2012; Lam et al., 2008; Lam et al., 2011; Schultz-Cherry et al., 2001; Zhang et al., 2011). 2012; Lam et al., 2008; Lam et al., 2011; Schultz-Cherry et al., 2001; Zhang et al., 2011). The mechanism how NS1 induces apoptosis is not known. Zhang et al. (2011) proposed, The mechanism how NS1 induces apoptosis is not known. Zhang et al. (2011) proposed, that NS1 interacts with Hsp90 (heat shock protein 90), which would promote the that NS1 interacts with Hsp90 (heat shock protein 90), which would promote the activation of caspase cascade (Zhang et al., 2011). activation of caspase cascade (Zhang et al., 2011). It may be that controversial apoptosis mechanisms reported by NS1 are caused by the It may be that controversial apoptosis mechanisms reported by NS1 are caused by the differences in virus strain NS1 proteins, as well as the host systems used in the studies. differences in virus strain NS1 proteins, as well as the host systems used in the studies. It may also be that the differential apoptosis control is temporally regulated, so that It may also be that the differential apoptosis control is temporally regulated, so that apoptosis is inhibited early during infection (promotes the replication of the genome), apoptosis is inhibited early during infection (promotes the replication of the genome), whilst enhanced at late time points of infection (increases the release of progeny whilst enhanced at late time points of infection (increases the release of progeny virions). virions).

1.3.6 Interactions of NS1 with other host cell factors 1.3.6 Interactions of NS1 with other host cell factors

1.3.6.1 PDZ domain mediated interactions 1.3.6.1 PDZ domain mediated interactions The PDZ domains bind to a specific PDZ binding motif (PBM) that is typically found at the The PDZ domains bind to a specific PDZ binding motif (PBM) that is typically found at the extreme C-terminus of a target protein, although in some cases the binding motif can extreme C-terminus of a target protein, although in some cases the binding motif can have an internal location (Javier and Rice, 2011). The IAV NS1 protein displays PBM at have an internal location (Javier and Rice, 2011). The IAV NS1 protein displays PBM at the very C-terminus of the NS1 sequence (Obenauer et al., 2006). In avian IAV NS1 the very C-terminus of the NS1 sequence (Obenauer et al., 2006). In avian IAV NS1 protein the consensus sequence for PBM is either ESEV or EPEV, whereas in human IAV protein the consensus sequence for PBM is either ESEV or EPEV, whereas in human IAV isolates the consensus sequence is RSKV. Only the NS1 proteins from avian IAV isolates isolates the consensus sequence is RSKV. Only the NS1 proteins from avian IAV isolates have so far been reported to bind to PDZ domain containing proteins, which have anti- have so far been reported to bind to PDZ domain containing proteins, which have anti- apoptotic or cell polarity regulatory functions (Javier and Rice, 2011). The interaction of apoptotic or cell polarity regulatory functions (Javier and Rice, 2011). The interaction of NS1 PBM with two PDZ domains of Scribble protein protected the IAV infected cells from NS1 PBM with two PDZ domains of Scribble protein protected the IAV infected cells from apoptosis (Liu et al., 2010). Furthermore, the interaction of NS1 with PDZ domains of apoptosis (Liu et al., 2010). Furthermore, the interaction of NS1 with PDZ domains of

22 22 Scribble and Dlg-1 was reported to disrupt cellular tight junctions to increase the Scribble and Dlg-1 was reported to disrupt cellular tight junctions to increase the permeability of infected cells (Golebiewski et al., 2011). NS1 PBM also mediates the permeability of infected cells (Golebiewski et al., 2011). NS1 PBM also mediates the binding and inactivation of MAG-1, which leads to higher IFN-β production (Kumar et al., binding and inactivation of MAG-1, which leads to higher IFN-β production (Kumar et al., 2012). This rather unexpected function for NS1 seems to be masked by other IAV anti- 2012). This rather unexpected function for NS1 seems to be masked by other IAV anti- IFN functions and the relevance of this interaction to IAV infection remains to be solved. IFN functions and the relevance of this interaction to IAV infection remains to be solved. The ESEV-sequence was found to increase the replication and virulence of human The ESEV-sequence was found to increase the replication and virulence of human A/WSN (A/WSN/33) IAV strain (Jackson et al., 2008) and H3N2 IAV strains (Liu et al., A/WSN (A/WSN/33) IAV strain (Jackson et al., 2008) and H3N2 IAV strains (Liu et al., 2010) when introduced to the NS1 sequence of these viruses. In addition the avian H7N1 2010) when introduced to the NS1 sequence of these viruses. In addition the avian H7N1 strain was shown to benefit from the ESEV-sequence since the mutation of the motif strain was shown to benefit from the ESEV-sequence since the mutation of the motif resulted in lower replication of the virus (Soubies et al., 2010). In contrast, when the resulted in lower replication of the virus (Soubies et al., 2010). In contrast, when the ESEV-sequence was introduced into highly pathogenic H5N1 virus it did not affect ESEV-sequence was introduced into highly pathogenic H5N1 virus it did not affect virulence in a mouse model (Zielecki et al., 2010). Similarly, the deletion of PBM from virulence in a mouse model (Zielecki et al., 2010). Similarly, the deletion of PBM from NS1 in low-pathogenic avian H7N1 virus strains had no effect on replication (Soubies et NS1 in low-pathogenic avian H7N1 virus strains had no effect on replication (Soubies et al., 2013). Likewise, the restoration of PBM in the NS1 of the 2009 H1N1 pandemic virus al., 2013). Likewise, the restoration of PBM in the NS1 of the 2009 H1N1 pandemic virus did not have an effect on the viral growth in cultured cells (Hale et al., 2010b) but had a did not have an effect on the viral growth in cultured cells (Hale et al., 2010b) but had a positive effect on viral transmission in a guinea pig model (Kim et al., 2014). Thus, the positive effect on viral transmission in a guinea pig model (Kim et al., 2014). Thus, the requirement of PBM for viral replication is strictly virus strain specific. requirement of PBM for viral replication is strictly virus strain specific.

1.3.6.2 SH3 domain mediated interactions 1.3.6.2 SH3 domain mediated interactions The Src homology 3 (SH3) domains bind to proline-rich sequences to transmit various The Src homology 3 (SH3) domains bind to proline-rich sequences to transmit various signals (see below). As first reported in study I of this thesis, the Spanish Flu and many signals (see below). As first reported in study I of this thesis, the Spanish Flu and many avian IAV NS1 proteins contain a functional SH3 domain binding motif close to their C- avian IAV NS1 proteins contain a functional SH3 domain binding motif close to their C- terminus. This motif mediates the binding of NS1 to the SH3 domains of Crk adaptor terminus. This motif mediates the binding of NS1 to the SH3 domains of Crk adaptor proteins. The NS1 proteins of human IAVs do not possess this motif. Hrincius et al. proteins. The NS1 proteins of human IAVs do not possess this motif. Hrincius et al. showed that downregulation of Crk proteins resulted in impaired propagation of IAVs showed that downregulation of Crk proteins resulted in impaired propagation of IAVs that contain SH3 binding competent NS1 protein due to the inhibition of IAV-mediated that contain SH3 binding competent NS1 protein due to the inhibition of IAV-mediated activation of c-jun N-terminal kinase (JNK) signaling pathway (Hrincius et al., 2010). activation of c-jun N-terminal kinase (JNK) signaling pathway (Hrincius et al., 2010). Especially, CrkI was shown to be important for the inhibition of JNK-pathway and Especially, CrkI was shown to be important for the inhibition of JNK-pathway and subsequent inhibition of apoptosis. In another study by the same group, NS1 was subsequent inhibition of apoptosis. In another study by the same group, NS1 was demonstrated to reduce the activity of tyrosine kinase c-Abl (Hrincius et al., 2014). The demonstrated to reduce the activity of tyrosine kinase c-Abl (Hrincius et al., 2014). The inhibition was shown to be mediated by the Crk-binding capacity of NS1 but also by a inhibition was shown to be mediated by the Crk-binding capacity of NS1 but also by a direct interaction of NS1 with c-Abl. The reduced activity of c-Abl led to lower basal direct interaction of NS1 with c-Abl. The reduced activity of c-Abl led to lower basal levels of Crk phosphorylation and severe cytopathic effects of infected cells. Chemical levels of Crk phosphorylation and severe cytopathic effects of infected cells. Chemical inhibition of c-Abl impaired propagation and pathogenicity of avian IAV. The NS1- inhibition of c-Abl impaired propagation and pathogenicity of avian IAV. The NS1- mediated inhibition of c-Abl was further shown to result in severe lung injury and to mediated inhibition of c-Abl was further shown to result in severe lung injury and to facilitate secondary bacterial infections in mice when functional SH3 binding motif was facilitate secondary bacterial infections in mice when functional SH3 binding motif was introduced into the NS1 protein of human IAV strain A/PR8 (Hrincius et al., 2015). introduced into the NS1 protein of human IAV strain A/PR8 (Hrincius et al., 2015).

1.3.7 Regulation of viral RNA and protein synthesis by NS1 1.3.7 Regulation of viral RNA and protein synthesis by NS1 The gene expression of IAV can be divided into an early and late phase. NS and NP vRNAs The gene expression of IAV can be divided into an early and late phase. NS and NP vRNAs are replicated and transcribed in the early phase, while in the late phase all 8 vRNAs are are replicated and transcribed in the early phase, while in the late phase all 8 vRNAs are replicated and transcribed (Shapiro et al., 1987; Skehel, 1973). Several studies have replicated and transcribed (Shapiro et al., 1987; Skehel, 1973). Several studies have provided evidence that NS1 is involved in the regulation of vRNA synthesis. Mutation in provided evidence that NS1 is involved in the regulation of vRNA synthesis. Mutation in the NS1 sequence at residues 123 and 124 resulted in deregulation of the normal time the NS1 sequence at residues 123 and 124 resulted in deregulation of the normal time course of vRNA synthesis, with both early and late vRNAs being replicated and course of vRNA synthesis, with both early and late vRNAs being replicated and transcribed at high levels already at very early times after infection (Min et al., 2007), transcribed at high levels already at very early times after infection (Min et al., 2007), while deletion of whole ED impaired the transcription of late genes (Maamary et al., while deletion of whole ED impaired the transcription of late genes (Maamary et al.,

23 23 2012). The mechanism how NS1 regulates temporally vRNA synthesis is not known. It 2012). The mechanism how NS1 regulates temporally vRNA synthesis is not known. It has been suggested that the interaction of NS1 with virus polymerase complex plays a has been suggested that the interaction of NS1 with virus polymerase complex plays a role in the process (Kuo and Krug, 2009). In addition, a recent study by Chen et al. role in the process (Kuo and Krug, 2009). In addition, a recent study by Chen et al. suggested that the inhibition of DDX21, a helicase able to block the viral polymerase suggested that the inhibition of DDX21, a helicase able to block the viral polymerase complex, by NS1 may be the key factor for this regulation (Chen et al., 2014). complex, by NS1 may be the key factor for this regulation (Chen et al., 2014). In IAV infected cells, the translation of host cell mRNAs is strongly inhibited, so the viral In IAV infected cells, the translation of host cell mRNAs is strongly inhibited, so the viral proteins may be translated efficiently (Garfinkel and Katze, 1993). NS1 has also shown proteins may be translated efficiently (Garfinkel and Katze, 1993). NS1 has also shown to stimulate the synthesis of viral proteins (de la Luna et al., 1995). Amino acids at to stimulate the synthesis of viral proteins (de la Luna et al., 1995). Amino acids at positions 25/26, 48, and 67 in the RBD of NS1 have been reported to be essential for the positions 25/26, 48, and 67 in the RBD of NS1 have been reported to be essential for the stimulation of translation (Kainov et al., 2011). The NS1 binds to 5’ untranslated region stimulation of translation (Kainov et al., 2011). The NS1 binds to 5’ untranslated region of viral mRNA (Park and Katze, 1995) and to several proteins involved in eukaryotic of viral mRNA (Park and Katze, 1995) and to several proteins involved in eukaryotic translation, such as eIF4GI (elongation and initiation factor 4GI) (Aragon et al., 2000) and translation, such as eIF4GI (elongation and initiation factor 4GI) (Aragon et al., 2000) and PABP1 (Burgui et al., 2003), as well as hStaufen (Falcon et al., 1999), a dsRNA binding PABP1 (Burgui et al., 2003), as well as hStaufen (Falcon et al., 1999), a dsRNA binding protein involved in the transport of mRNA to activate translation sites. The proposed protein involved in the transport of mRNA to activate translation sites. The proposed model is that NS1 recruits these translation factors to the 5’ ends of viral mRNAs to favor model is that NS1 recruits these translation factors to the 5’ ends of viral mRNAs to favor the translation of viral proteins. the translation of viral proteins.

1.4 PI3K/Akt pathway 1.4 PI3K/Akt pathway

1.4.1 An overview of the PI3K/Akt pathway 1.4.1 An overview of the PI3K/Akt pathway The phosphatidylinositol-3-kinases (PI3Ks) are members of a conserved family of The phosphatidylinositol-3-kinases (PI3Ks) are members of a conserved family of intracellular kinases that exhibit both protein kinase and lipid kinase activity. They are intracellular kinases that exhibit both protein kinase and lipid kinase activity. They are classified in three main classes (I-III) according to their substrate specificity and classified in three main classes (I-III) according to their substrate specificity and sequence homology. Class I PI3Ks are further divided into two subclasses: IA and IB. Class sequence homology. Class I PI3Ks are further divided into two subclasses: IA and IB. Class IA PI3Ks are heterodimers that consist of a regulatory subunit and a catalytic subunit. In IA PI3Ks are heterodimers that consist of a regulatory subunit and a catalytic subunit. In mammalians there are three class IA catalytic (p110 α, β and δ) and five regulatory mammalians there are three class IA catalytic (p110 α, β and δ) and five regulatory (p85α, p85β, p55γ, p55α, and p50α) subunits known (Engelman et al., 2006). Cellular (p85α, p85β, p55γ, p55α, and p50α) subunits known (Engelman et al., 2006). Cellular responses that involve PI3K signaling include survival, proliferation, trafficking, and responses that involve PI3K signaling include survival, proliferation, trafficking, and regulation of immune function. Basal activity of PI3K/Akt signaling ensures cell survival, regulation of immune function. Basal activity of PI3K/Akt signaling ensures cell survival, while inactivation of the pathway results in apoptosis. while inactivation of the pathway results in apoptosis. The activation of PI3K is tightly controlled (see Figure 4 for schematic representation of The activation of PI3K is tightly controlled (see Figure 4 for schematic representation of activation of PI3K/Akt signaling). The PI3K pathway is activated upon ligand binding on activation of PI3K/Akt signaling). The PI3K pathway is activated upon ligand binding on cell surface receptor tyrosine kinases (RTKs) which leads to dimerization and cell surface receptor tyrosine kinases (RTKs) which leads to dimerization and autophosphorylation of RTKs. The regulatory subunit p85 contains an SH2 (Src- autophosphorylation of RTKs. The regulatory subunit p85 contains an SH2 (Src- homology 2) domain which binds to tyrosine phosphorylated YXXM motifs on the RTKs. homology 2) domain which binds to tyrosine phosphorylated YXXM motifs on the RTKs. This triggers the activation of p110 catalytic subunit leading to conversion of This triggers the activation of p110 catalytic subunit leading to conversion of phosphatidylinositol (3,4)-bisphosphate (PIP2) lipids to phosphatidylinositol (3,4,5)- phosphatidylinositol (3,4)-bisphosphate (PIP2) lipids to phosphatidylinositol (3,4,5)- trisphosphate (PIP3). PIP3 serves as a second messenger interacting with PH (pleckstrin trisphosphate (PIP3). PIP3 serves as a second messenger interacting with PH (pleckstrin homology) domain-containing proteins. PI3K has several downstream signaling homology) domain-containing proteins. PI3K has several downstream signaling mediators. One important mediator is Akt (also known as PKB; protein kinase B) that mediators. One important mediator is Akt (also known as PKB; protein kinase B) that binds to PIP3, allowing PDK-1 (phosphoinositide-dependent kinase-1) to phosphorylate binds to PIP3, allowing PDK-1 (phosphoinositide-dependent kinase-1) to phosphorylate threonine at the position 308 (T308) on Akt. The full activation of Akt requires another threonine at the position 308 (T308) on Akt. The full activation of Akt requires another phosphorylation of serine at position 473 (S473). The kinase responsible for S473 phosphorylation of serine at position 473 (S473). The kinase responsible for S473 phosphorylation is either mTORC2 (mammalian target of rapamycin complex 2) phosphorylation is either mTORC2 (mammalian target of rapamycin complex 2) (Sarbassov et al., 2005) or DNA-PK (DNA-dependent protein kinase) (Feng et al., 2004) (Sarbassov et al., 2005) or DNA-PK (DNA-dependent protein kinase) (Feng et al., 2004) depending on the initial stimulus. depending on the initial stimulus.

24 24 Akt is a serine/threonine kinase which upon full activation phosphorylates many target Akt is a serine/threonine kinase which upon full activation phosphorylates many target proteins (Engelman et al., 2006). Regulation of apoptosis is an important function proteins (Engelman et al., 2006). Regulation of apoptosis is an important function ascribed to PI3K/Akt -signaling. Akt promotes cell survival by phosphorylating and ascribed to PI3K/Akt -signaling. Akt promotes cell survival by phosphorylating and inactivating pro-apoptotic proteins, such as FasL (Fas-ligand), BAD (Bcl2-antagonist of inactivating pro-apoptotic proteins, such as FasL (Fas-ligand), BAD (Bcl2-antagonist of cell death) and caspase-9. Active Akt is also involved in other cellular functions, including cell death) and caspase-9. Active Akt is also involved in other cellular functions, including angiogenesis, metabolism, growth, protein synthesis, proliferation, and survival. angiogenesis, metabolism, growth, protein synthesis, proliferation, and survival. PI3K/Akt signaling is negatively regulated by different phosphatases. Dephosphorylation PI3K/Akt signaling is negatively regulated by different phosphatases. Dephosphorylation of Akt is mediated by phosphatases PP2A (protein phosphatase 2A) (Millward et al., of Akt is mediated by phosphatases PP2A (protein phosphatase 2A) (Millward et al., 1999), and PHLPP (PH domain and leucine-rich repeat protein phosphatase) (Gao et al., 1999), and PHLPP (PH domain and leucine-rich repeat protein phosphatase) (Gao et al., 2005). The most important negative regulator of PI3K signaling is the PTEN (phosphatase 2005). The most important negative regulator of PI3K signaling is the PTEN (phosphatase of tensin homologue deleted on chromosome 10) which dephosphorylates PIP3 back to of tensin homologue deleted on chromosome 10) which dephosphorylates PIP3 back to PIP2 (Lam et al., 2008; Lam et al., 2011; Stambolic et al., 1998). PIP2 (Lam et al., 2008; Lam et al., 2011; Stambolic et al., 1998).

Figure 4. Schematic representation of PI3K/Akt pathway activation. See the text for description. After Figure 4. Schematic representation of PI3K/Akt pathway activation. See the text for description. After Engelman et al., 2006; Hemmings and Restuccia, 2015. Engelman et al., 2006; Hemmings and Restuccia, 2015.

1.4.2 Targeting of the PI3K/Akt pathway by viruses 1.4.2 Targeting of the PI3K/Akt pathway by viruses Since the host cell PI3K/Akt signaling pathway has a critical regulatory role in many Since the host cell PI3K/Akt signaling pathway has a critical regulatory role in many cellular processes, including survival, proliferation, RNA processing, translation, and cellular processes, including survival, proliferation, RNA processing, translation, and regulation of immune function, viruses have evolved widely varying ways to target it regulation of immune function, viruses have evolved widely varying ways to target it (Diehl and Schaal, 2013; Engelman et al., 2006). Many enveloped viruses activate the (Diehl and Schaal, 2013; Engelman et al., 2006). Many enveloped viruses activate the PI3K/Akt pathway to facilitate the endocytic uptake of virions. For example, the PI3K/Akt pathway to facilitate the endocytic uptake of virions. For example, the activation of PI3K during endocytic uptake of Ebola viruses is required since the activation of PI3K during endocytic uptake of Ebola viruses is required since the inhibition of the pathway leads to suppression of virus infection at an early step (Saeed inhibition of the pathway leads to suppression of virus infection at an early step (Saeed et al., 2008). Vaccinia virus, herpes simplex virus type-1 and IAV also requires PI3K et al., 2008). Vaccinia virus, herpes simplex virus type-1 and IAV also requires PI3K activation for host cell uptake (Diehl and Schaal, 2013; Ehrhardt et al., 2006). activation for host cell uptake (Diehl and Schaal, 2013; Ehrhardt et al., 2006). In addition to entry, many viruses manipulate PI3K/Akt signaling to control apoptosis In addition to entry, many viruses manipulate PI3K/Akt signaling to control apoptosis during the viral infection (Diehl and Schaal, 2013). The viral protein LMP1 (latent during the viral infection (Diehl and Schaal, 2013). The viral protein LMP1 (latent membrane protein 1) of Epstein-Barr (EBV) virus activates the PI3K/Akt pathway by membrane protein 1) of Epstein-Barr (EBV) virus activates the PI3K/Akt pathway by direct interaction with the p85 subunit, resulting in cell survival (Dawson et al., 2003). direct interaction with the p85 subunit, resulting in cell survival (Dawson et al., 2003). Rotavirus NSP1 and hepatitis C virus (HCV) NS5A (nonstructural protein 5A) also interact Rotavirus NSP1 and hepatitis C virus (HCV) NS5A (nonstructural protein 5A) also interact with p85 to induce PI3K/Akt pathway (Bagchi et al., 2010; Street et al., 2004). NS5A binds with p85 to induce PI3K/Akt pathway (Bagchi et al., 2010; Street et al., 2004). NS5A binds

25 25 to p85 and the interaction is mediated by the SH3 domain of p85. In addition, herpes to p85 and the interaction is mediated by the SH3 domain of p85. In addition, herpes simplex virus-1, respiratory syncytial virus, coxsackie virus B3, rubella virus, HIV-1, and simplex virus-1, respiratory syncytial virus, coxsackie virus B3, rubella virus, HIV-1, and IAV prevents apoptosis by inducing the PI3K/Akt pathway during infection (Diehl and IAV prevents apoptosis by inducing the PI3K/Akt pathway during infection (Diehl and Schaal, 2013). Some viruses utilize the PI3K/Akt pathway to regulate the translation of Schaal, 2013). Some viruses utilize the PI3K/Akt pathway to regulate the translation of viral proteins by activating mTOR (mammalian target of rapamycin), a downstream viral proteins by activating mTOR (mammalian target of rapamycin), a downstream target of Akt. EBV LMP2A, adenovirus E4-ORF1, and E6 and E7 proteins of human target of Akt. EBV LMP2A, adenovirus E4-ORF1, and E6 and E7 proteins of human papilloma virus are examples of viral proteins which have been reported to activate papilloma virus are examples of viral proteins which have been reported to activate mTOR during infection (Diehl and Schaal, 2013). mTOR during infection (Diehl and Schaal, 2013).

1.4.3 Induction of the PI3K/Akt pathway by NS1 protein 1.4.3 Induction of the PI3K/Akt pathway by NS1 protein The PI3K signaling is activated twice in IAV infected cells (Ehrhardt et al., 2006). The The PI3K signaling is activated twice in IAV infected cells (Ehrhardt et al., 2006). The attachment and entry of the virus cause the first, a transient PI3K activation, which is attachment and entry of the virus cause the first, a transient PI3K activation, which is related to the uptake of virus by endocytosis. The second wave of activation, sustained related to the uptake of virus by endocytosis. The second wave of activation, sustained activation, appears 4-6 hours post-infection (Ehrhardt et al., 2006; Hale et al., 2006). The activation, appears 4-6 hours post-infection (Ehrhardt et al., 2006; Hale et al., 2006). The latter activation was abolished when an IAV lacking NS1 (delNS1) was used (Ehrhardt et latter activation was abolished when an IAV lacking NS1 (delNS1) was used (Ehrhardt et al., 2006). In addition, stable expression of NS1 alone was able to induce the al., 2006). In addition, stable expression of NS1 alone was able to induce the phosphorylation of Akt at S473, which was sensitive to general PI3K inhibitor (Hale et phosphorylation of Akt at S473, which was sensitive to general PI3K inhibitor (Hale et al., 2006). Thus, the second activation of PI3K in IAV infected cells is mediated by NS1. al., 2006). Thus, the second activation of PI3K in IAV infected cells is mediated by NS1. The structural and mechanistic basis for PI3K activation by NS1 has been well studied. It The structural and mechanistic basis for PI3K activation by NS1 has been well studied. It is mediated by the interaction of NS1 with the p85 regulatory subunit of PI3K. The is mediated by the interaction of NS1 with the p85 regulatory subunit of PI3K. The interaction is isoform specific, since NS1 interacts only with the β isoform of p85 (Hale interaction is isoform specific, since NS1 interacts only with the β isoform of p85 (Hale et al., 2006; Shin et al., 2007a). The interaction preface between NS1 and p85β has been et al., 2006; Shin et al., 2007a). The interaction preface between NS1 and p85β has been established by mutational analyses as well as by resolving the crystal structure of the established by mutational analyses as well as by resolving the crystal structure of the interaction (Hale et al., 2008b; Hale et al., 2006; Hale et al., 2010a; Li et al., 2008). interaction (Hale et al., 2008b; Hale et al., 2006; Hale et al., 2010a; Li et al., 2008). p85β contains five domains: an SH3-domain, a GTPase activating , two p85β contains five domains: an SH3-domain, a GTPase activating protein domain, two SH2-domains, and an inter SH2 (iSH2) domain which separates the two SH2 domains SH2-domains, and an inter SH2 (iSH2) domain which separates the two SH2 domains (Okkenhaug and Vanhaesebroeck, 2001). The p85 subunit binds to p110 through the (Okkenhaug and Vanhaesebroeck, 2001). The p85 subunit binds to p110 through the iSH2 domain (Dhand et al., 1994; Klippel et al., 1993) where NS1 also binds (Hale et al., iSH2 domain (Dhand et al., 1994; Klippel et al., 1993) where NS1 also binds (Hale et al., 2008b). Tyrosine residue at the position of 89 (Y89) has been reported to be crucial for 2008b). Tyrosine residue at the position of 89 (Y89) has been reported to be crucial for the interaction and substitution of tyrosine at this position to phenylalanine (Y89F) the interaction and substitution of tyrosine at this position to phenylalanine (Y89F) abrogates the NS1-p85β binding and PI3K/Akt activation (Hale et al., 2006). The crystal abrogates the NS1-p85β binding and PI3K/Akt activation (Hale et al., 2006). The crystal structure of the interaction confirmed that the residue Y89 and a proline at 164 (P164) structure of the interaction confirmed that the residue Y89 and a proline at 164 (P164) are located at the binding interface with p85β (Hale et al., 2010b). Glutamine residue are located at the binding interface with p85β (Hale et al., 2010b). Glutamine residue 142 in NS1 seems to also contribute for the interaction (Hale et al., 2010b; Li et al., 2008). 142 in NS1 seems to also contribute for the interaction (Hale et al., 2010b; Li et al., 2008). In p85β the crucial amino acid for the interaction is valine at position 573 (Li et al., 2008). In p85β the crucial amino acid for the interaction is valine at position 573 (Li et al., 2008). Mutation of the corresponding residue on p85α (methionine 582) to valine enables NS1 Mutation of the corresponding residue on p85α (methionine 582) to valine enables NS1 to interact also with p85α (Li et al., 2008). Crystal structure of the interaction reveals to interact also with p85α (Li et al., 2008). Crystal structure of the interaction reveals that methionine instead of valine at this position in p85β would be sterically precluded, that methionine instead of valine at this position in p85β would be sterically precluded, explaining the strict isoform specificity of NS1-p85 binding (Hale et al., 2010a). The explaining the strict isoform specificity of NS1-p85 binding (Hale et al., 2010a). The association of NS1 with p85β removes the inhibitory contacts between the p85β and association of NS1 with p85β removes the inhibitory contacts between the p85β and p110 leading to the activation of PI3K (Hale et al., 2008b; Hale et al., 2010a). In addition, p110 leading to the activation of PI3K (Hale et al., 2008b; Hale et al., 2010a). In addition, NS1 possibly directly stimulates the activity of p110 (Hale et al., 2010b). Mutations in a NS1 possibly directly stimulates the activity of p110 (Hale et al., 2010b). Mutations in a small, acidic α-helix in NS1 (residues 95-100) that lies adjacent to the activation loop of small, acidic α-helix in NS1 (residues 95-100) that lies adjacent to the activation loop of p110, generated a virus that was unable to produce PIP3 during the infection, and p110, generated a virus that was unable to produce PIP3 during the infection, and consequently failed to induce S473 phosphorylation of Akt. consequently failed to induce S473 phosphorylation of Akt.

26 26 The biological relevance of NS1-mediated PI3K activation for IAV infection is still not The biological relevance of NS1-mediated PI3K activation for IAV infection is still not clear. As the PI3K signaling is known to mediate cell survival via activation of Akt (Cooray, clear. As the PI3K signaling is known to mediate cell survival via activation of Akt (Cooray, 2004), the NS1-mediated PI3K activation has been proposed to stimulate cell survival. 2004), the NS1-mediated PI3K activation has been proposed to stimulate cell survival. By using chemical PI3K-inhibitors during virus infection, NS1-mediated PI3K activation By using chemical PI3K-inhibitors during virus infection, NS1-mediated PI3K activation was reported to be involved in inhibition of apoptosis in a human A/PR8 and an avian was reported to be involved in inhibition of apoptosis in a human A/PR8 and an avian A/FPV/Bratislava/79 IAV strains (Ehrhardt et al., 2007; Zhirnov and Klenk, 2007). A/FPV/Bratislava/79 IAV strains (Ehrhardt et al., 2007; Zhirnov and Klenk, 2007). Likewise, Shin et al. reported a more proapoptotic phenotype of A/PR8 IAV with an NS1 Likewise, Shin et al. reported a more proapoptotic phenotype of A/PR8 IAV with an NS1 mutated at residue P164, which is important for p85-binding (Shin et al., 2007a). Several mutated at residue P164, which is important for p85-binding (Shin et al., 2007a). Several studies have also reported the importance of NS1-mediated PI3K activation for viral studies have also reported the importance of NS1-mediated PI3K activation for viral replication and virulence. Ehrhardt and colleagues used a chemical PI3K-inhibitor during replication and virulence. Ehrhardt and colleagues used a chemical PI3K-inhibitor during the infection of cultured cells with A/PR8 and A/FPV IAV strains (Ehrhardt et al., 2006). the infection of cultured cells with A/PR8 and A/FPV IAV strains (Ehrhardt et al., 2006). The inhibition of PI3K resulted in impaired viral propagation. Likewise, by mutating the The inhibition of PI3K resulted in impaired viral propagation. Likewise, by mutating the NS1 residue Y89 in human A/PR8 virus, it was shown that activation of PI3K signaling NS1 residue Y89 in human A/PR8 virus, it was shown that activation of PI3K signaling enhances viral replication in tissue culture and the virulence in a mouse model (Ayllon enhances viral replication in tissue culture and the virulence in a mouse model (Ayllon et al., 2012b; Hrincius et al., 2012). However, the requirement for PI3K activation seems et al., 2012b; Hrincius et al., 2012). However, the requirement for PI3K activation seems to be viral strain specific, since the Y89F mutation in another human IAV strain, A/WSN, to be viral strain specific, since the Y89F mutation in another human IAV strain, A/WSN, did not have any effect on viral replication or virulence (Ayllon et al., 2012b). did not have any effect on viral replication or virulence (Ayllon et al., 2012b). Furthermore, mutation in A/Udorn NS1 to block the interaction with p85β and Furthermore, mutation in A/Udorn NS1 to block the interaction with p85β and subsequent loss of PI3K activation had no effect on IAV infected cell viability (Jackson et subsequent loss of PI3K activation had no effect on IAV infected cell viability (Jackson et al., 2010). al., 2010).

1.5 SH3 domains 1.5 SH3 domains The SH3 domains are short, approximately 50-70 residues long, and they are The SH3 domains are short, approximately 50-70 residues long, and they are characterized by their ability to bind to proline-rich peptide modules. They were characterized by their ability to bind to proline-rich peptide modules. They were discovered in 1988 when two groups identified independent regions of sequence discovered in 1988 when two groups identified independent regions of sequence similarity between divergent signaling proteins of the Src family of non-receptor similarity between divergent signaling proteins of the Src family of non-receptor tyrosine kinases, the Crk oncogene, and phospholipase C-γ (PLCγ) (Mayer et al., 1988; tyrosine kinases, the Crk oncogene, and phospholipase C-γ (PLCγ) (Mayer et al., 1988; Stahl et al., 1988). The fact that the domain was found in many different proteins and Stahl et al., 1988). The fact that the domain was found in many different proteins and did not appear to have any enzymatic activity, implied that the domain was modular and did not appear to have any enzymatic activity, implied that the domain was modular and possessed independent functions in protein complexes. possessed independent functions in protein complexes. SH3 domains are one of the most common modular protein domains found in eukaryotic SH3 domains are one of the most common modular protein domains found in eukaryotic genomes (Mayer and Saksela, 2004). The human genome has been estimated to contain genomes (Mayer and Saksela, 2004). The human genome has been estimated to contain 296 different SH3 domains (Kärkkäinen et al., 2006). Since one protein may contain up 296 different SH3 domains (Kärkkäinen et al., 2006). Since one protein may contain up to six SH3 domains, the number for SH3 domain containing proteins is somewhat to six SH3 domains, the number for SH3 domain containing proteins is somewhat smaller. SH3 domains are present in many different cellular proteins, including scaffold smaller. SH3 domains are present in many different cellular proteins, including scaffold proteins, adapter proteins and enzymes. They mediate specific protein-protein proteins, adapter proteins and enzymes. They mediate specific protein-protein interactions and they are involved in many important cellular functions, such as interactions and they are involved in many important cellular functions, such as regulation of signal transduction, cytoskeletal organization and membrane trafficking regulation of signal transduction, cytoskeletal organization and membrane trafficking (Mayer and Saksela, 2004). Binding of an SH3 domain to its specific ligand recruits (Mayer and Saksela, 2004). Binding of an SH3 domain to its specific ligand recruits proteins to various subcellular locations, assembles multi-protein signaling complexes, proteins to various subcellular locations, assembles multi-protein signaling complexes, and regulates enzyme activities. Many SH3 domain containing proteins also possess and regulates enzyme activities. Many SH3 domain containing proteins also possess other protein-protein interaction domains, such as SH2 (Src homology 2) domains, other protein-protein interaction domains, such as SH2 (Src homology 2) domains, emphasizing the importance of these proteins in multi-protein complex formation and emphasizing the importance of these proteins in multi-protein complex formation and signal transduction in cells. SH2 domains bind to short phosphotyrosine containing signal transduction in cells. SH2 domains bind to short phosphotyrosine containing motifs within their target proteins to mediate similar functions as SH3 domains. motifs within their target proteins to mediate similar functions as SH3 domains.

27 27 1.5.1 SH3 domain structure 1.5.1 SH3 domain structure Crystallography and NMR studies have revealed the highly conserved three dimensional Crystallography and NMR studies have revealed the highly conserved three dimensional structure of SH3 domains (Musacchio et al., 1992; Yu et al., 1992). They consist of five structure of SH3 domains (Musacchio et al., 1992; Yu et al., 1992). They consist of five β-strands (β1- β5) that are arranged to two tightly packed anti-parallel β-sheets. The first β-strands (β1- β5) that are arranged to two tightly packed anti-parallel β-sheets. The first β-sheet is composed of β1, half of the β2 and the β5, while the second is formed of the β-sheet is composed of β1, half of the β2 and the β5, while the second is formed of the second half of the β2, β3 and β4 -strands. The β-strands are connected by three variable second half of the β2, β3 and β4 -strands. The β-strands are connected by three variable loops, denoted as RT, n-Src, and distal loops as well as by a short 3 10 –helix. The first two loops, denoted as RT, n-Src, and distal loops as well as by a short 3 10 –helix. The first two strands (β1- β2) are separated by the RT, the β2- β3 by the n-Src, the β3- β4 by the distal strands (β1- β2) are separated by the RT, the β2- β3 by the n-Src, the β3- β4 by the distal loops, and the last two (β4- β5) by the 310 –helix. loops, and the last two (β4- β5) by the 310 –helix. The ligand binding site of SH3 domain has a relatively shallow binding surface. It is The ligand binding site of SH3 domain has a relatively shallow binding surface. It is formed by the conserved hydrophobic residues in β3- and β4-strands, N-Src loop and the formed by the conserved hydrophobic residues in β3- and β4-strands, N-Src loop and the tip of the RT loop (Musacchio et al., 1992; Yu et al., 1992). The binding surface can be tip of the RT loop (Musacchio et al., 1992; Yu et al., 1992). The binding surface can be divided into three binding pockets: two hydrophobic pockets, lined mainly by aromatic divided into three binding pockets: two hydrophobic pockets, lined mainly by aromatic residues and a specificity pocket, formed by residues from RT and n-Src loops. residues and a specificity pocket, formed by residues from RT and n-Src loops.

1.5.2 SH3 domain ligand binding motifs 1.5.2 SH3 domain ligand binding motifs

1.5.2.1 Typical ligand binding motifs 1.5.2.1 Typical ligand binding motifs Early studies identified that the SH3 domains bind to proline-rich peptides (Ren et al., Early studies identified that the SH3 domains bind to proline-rich peptides (Ren et al., 1993). It was further noticed that SH3 domains bind specifically to two consensus 1993). It was further noticed that SH3 domains bind specifically to two consensus peptides containing ɸP moieties, where ɸ is usually a hydrophobic residue (Kay et al., peptides containing ɸP moieties, where ɸ is usually a hydrophobic residue (Kay et al., 2000). SH3 ligand peptide adopts a polyproline-2 (PPII) structure, a left handed helix, 2000). SH3 ligand peptide adopts a polyproline-2 (PPII) structure, a left handed helix, which has three residues per turn and is roughly triangular in cross-section (Musacchio, which has three residues per turn and is roughly triangular in cross-section (Musacchio, 2002). The base of this triangle sits on the surface of the SH3 domain and the two 2002). The base of this triangle sits on the surface of the SH3 domain and the two hydrophobic pockets of the SH3 binding site recognize ɸP dipeptides of the PPII helix, hydrophobic pockets of the SH3 binding site recognize ɸP dipeptides of the PPII helix, whereas the third, “specificity” pocket binds to a positively charged residue, which whereas the third, “specificity” pocket binds to a positively charged residue, which flanks the ɸPxɸP core binding motif (see Figure 5). The positively charged residue is flanks the ɸPxɸP core binding motif (see Figure 5). The positively charged residue is typically either lysine (K) or more commonly arginine (R). typically either lysine (K) or more commonly arginine (R). The structural studies on SH3-peptide complexes have identified that PPII helix can bind The structural studies on SH3-peptide complexes have identified that PPII helix can bind in two orientations with respect to the SH3 domain (Feng et al., 1994; Lim et al., 1994). in two orientations with respect to the SH3 domain (Feng et al., 1994; Lim et al., 1994). The peptides that can bind in N to C orientation relative to SH3 domain have been The peptides that can bind in N to C orientation relative to SH3 domain have been classified as class I ligands, whereas the C to N binding peptides are called class II ligands. classified as class I ligands, whereas the C to N binding peptides are called class II ligands. The orientation of the peptide depends on the location of a positively charged residue. The orientation of the peptide depends on the location of a positively charged residue. The consensus sequence for class I ligands is +xɸPxɸP, and for class II ligands ɸPxɸPx+ The consensus sequence for class I ligands is +xɸPxɸP, and for class II ligands ɸPxɸPx+ (where P is proline, + is R or K, ɸ is a hydrophobic residue, and x is any amino acid). Some (where P is proline, + is R or K, ɸ is a hydrophobic residue, and x is any amino acid). Some SH3 domains bind to the ligand peptide only in one orientation, whereas some can bind SH3 domains bind to the ligand peptide only in one orientation, whereas some can bind ligands in both orientations. ligands in both orientations.

28 28 Figure 5. Binding of class I and class II consensus ligands to SH3 domains. After Zarrinpar et al., 2003. Figure 5. Binding of class I and class II consensus ligands to SH3 domains. After Zarrinpar et al., 2003.

1.5.2.2 Atypical ligand binding motifs 1.5.2.2 Atypical ligand binding motifs In addition to class I and class II SH3 binding sites, a number of alternative motifs have In addition to class I and class II SH3 binding sites, a number of alternative motifs have been identified that lack the core PxxP element (Li, 2005; Mayer and Saksela, 2004). For been identified that lack the core PxxP element (Li, 2005; Mayer and Saksela, 2004). For example, the SH3 domains of Eps8 family members and the N-terminal SH3 domain of example, the SH3 domains of Eps8 family members and the N-terminal SH3 domain of Nck1 bind to ligands that do not contain canonical PxxP-motifs. Instead, they have a Nck1 bind to ligands that do not contain canonical PxxP-motifs. Instead, they have a unique binding preference for PxxDY motif (Kesti et al., 2007; Mongiovi et al., 1999). The unique binding preference for PxxDY motif (Kesti et al., 2007; Mongiovi et al., 1999). The first hydrophobic pocket of Eps8L1 SH3 domain is not optimal for binding the first hydrophobic pocket of Eps8L1 SH3 domain is not optimal for binding the conventional PxxP peptides since it is smaller than usually (Aitio et al., 2008). Some SH3 conventional PxxP peptides since it is smaller than usually (Aitio et al., 2008). Some SH3 binding motifs even totally lack the proline residues, like the SH3 domains of Fyn and binding motifs even totally lack the proline residues, like the SH3 domains of Fyn and Fyb, which bind selectively to RKxxYxxY consensus in SKAP55 (Src kinase-associated Fyb, which bind selectively to RKxxYxxY consensus in SKAP55 (Src kinase-associated protein of 55 kDa) adaptor protein (Kang et al., 2000). Also, SH3 domains of Fyn and protein of 55 kDa) adaptor protein (Kang et al., 2000). Also, SH3 domains of Fyn and Lck that bind to class I binding motifs, could interact with this motif. Hence, the recognition that bind to class I binding motifs, could interact with this motif. Hence, the recognition of RKxxYxxY seems to be similar to the class I consensus recognition sequence. of RKxxYxxY seems to be similar to the class I consensus recognition sequence. Moreover, several SH3 ligand proteins have been found to contain an R/KxxK/R Moreover, several SH3 ligand proteins have been found to contain an R/KxxK/R consensus binding site. For example, the C-terminal SH3 domains of Grb2 and Gads consensus binding site. For example, the C-terminal SH3 domains of Grb2 and Gads adaptor proteins interact with proteins that has the R/KxxK/R consensus (Berry et al., adaptor proteins interact with proteins that has the R/KxxK/R consensus (Berry et al., 2002; Lewitzky et al., 2001; Liu et al., 2003). The R/KxxK/R consensus is sometimes 2002; Lewitzky et al., 2001; Liu et al., 2003). The R/KxxK/R consensus is sometimes referred as the class III binding motif, since it is found in numerous SH3 ligand proteins referred as the class III binding motif, since it is found in numerous SH3 ligand proteins (Li, 2005). Many more unconventional binding site sequences have been identified, (Li, 2005). Many more unconventional binding site sequences have been identified, including the RxxPxxxxP consensus found in the calcium activated potassium channel including the RxxPxxxxP consensus found in the calcium activated potassium channel which mediates binding to the SH3 domain of (Tian et al., 2006). which mediates binding to the SH3 domain of cortactin (Tian et al., 2006).

1.5.3 Affinity and specificity of SH3 domains 1.5.3 Affinity and specificity of SH3 domains The affinity and specificity of SH3 domains to their ligands is relatively low and the The affinity and specificity of SH3 domains to their ligands is relatively low and the dissociation constant (Kd) normally varies in the 1-200 μM range (Mayer and Saksela, dissociation constant (Kd) normally varies in the 1-200 μM range (Mayer and Saksela, 2004). Since only five residues of the core binding motif have direct contact with the SH3 2004). Since only five residues of the core binding motif have direct contact with the SH3 domain, additional contacts between the variable loops of the SH3 domain and residues domain, additional contacts between the variable loops of the SH3 domain and residues outside the core PxxP motif in the ligand play a critical role in determining the specificity outside the core PxxP motif in the ligand play a critical role in determining the specificity and affinity of an SH3 domain. For example, the unusually high affinity (Kd 24 nM) of the and affinity of an SH3 domain. For example, the unusually high affinity (Kd 24 nM) of the

29 29 SH3 domain of p67phox phox (phagocyte oxidase) protein with p47phox requires 20 SH3 domain of p67phox phox (phagocyte oxidase) protein with p47phox requires 20 additional residues immediately C-terminal to the PxxP motif in p47phox (Kami et al., additional residues immediately C-terminal to the PxxP motif in p47phox (Kami et al., 2002). When the additional residues were removed, the affinity of the interaction was 2002). When the additional residues were removed, the affinity of the interaction was turned into a very modest one (Kd 20 μM). Also the avid and highly selective binding of turned into a very modest one (Kd 20 μM). Also the avid and highly selective binding of PEP (proline enriched phosphatase) to the SH3 domain of Csk requires additional PEP (proline enriched phosphatase) to the SH3 domain of Csk requires additional residues outside the binding motif. Two hydrophobic amino acids, isoleucine and valine, residues outside the binding motif. Two hydrophobic amino acids, isoleucine and valine, located six residues after the consensus class II PxxP motif in PEP are placed into the located six residues after the consensus class II PxxP motif in PEP are placed into the specificity pocket of Csk SH3 domain (Ghose et al., 2001). In addition, 310 helix structure specificity pocket of Csk SH3 domain (Ghose et al., 2001). In addition, 310 helix structure positioned after the PxxP motif in PEP helps to orientate the residues into the specificity positioned after the PxxP motif in PEP helps to orientate the residues into the specificity pocket. pocket. The binding of HIV-1 Nef protein with SH3 domain of Hck provides an The binding of HIV-1 Nef protein with SH3 domain of Src family kinase Hck provides an example of an SH3 domain interaction where the affinity and specificity both require example of an SH3 domain interaction where the affinity and specificity both require additional residues outside the PxxP motif in Nef in the ligand and sequence variation in additional residues outside the PxxP motif in Nef in the ligand and sequence variation in the RT-loop of an SH3 domain. The affinity of Hck SH3 domain to Nef is very high (K d 0.25 the RT-loop of an SH3 domain. The affinity of Hck SH3 domain to Nef is very high (K d 0.25 μM), whereas another Src family member, Fyn, has a very modest binding affinity (K d < μM), whereas another Src family member, Fyn, has a very modest binding affinity (K d < 20 μM) for Nef. It was found that a single amino acid at the tip of the RT loop of the Hck 20 μM) for Nef. It was found that a single amino acid at the tip of the RT loop of the Hck SH3 domain compared to Fyn was important for this high affinity binding (Lee et al., SH3 domain compared to Fyn was important for this high affinity binding (Lee et al., 1995). When the corresponding amino acid in the RT-loop of Fyn (arginine) was 1995). When the corresponding amino acid in the RT-loop of Fyn (arginine) was substituted for isoleucine as in Hck, the affinity of Fyn for Nef was almost as good as that substituted for isoleucine as in Hck, the affinity of Fyn for Nef was almost as good as that of Hck. In addition, the core PxxP site in Nef was not sufficient for the tight binding as a of Hck. In addition, the core PxxP site in Nef was not sufficient for the tight binding as a 12-residue long peptide overlapping the PxxP-site of Nef had only modest binding 12-residue long peptide overlapping the PxxP-site of Nef had only modest binding affinity and specificity towards Hck SH3, suggesting that additional regions in Nef are affinity and specificity towards Hck SH3, suggesting that additional regions in Nef are important for the interaction. The crystal structure of Nef-SH3 interaction revealed that important for the interaction. The crystal structure of Nef-SH3 interaction revealed that regions distal to the PxxP motif in Nef are important for the binding (Lee et al., 1996). regions distal to the PxxP motif in Nef are important for the binding (Lee et al., 1996). The PxxP motif of Nef is followed by two α-helices forming a hydrophobic pocket that The PxxP motif of Nef is followed by two α-helices forming a hydrophobic pocket that engages the specificity-determining isoleucine residue of the SH3 domain. engages the specificity-determining isoleucine residue of the SH3 domain.

1.5.4 Viral proteins as SH3 domain ligands 1.5.4 Viral proteins as SH3 domain ligands As SH3 domains are involved in regulation of numerous cellular events, they serve as a As SH3 domains are involved in regulation of numerous cellular events, they serve as a potential target for virus to regulate host cell signaling to optimize their replication potential target for virus to regulate host cell signaling to optimize their replication during the infection. Indeed, several viral proteins have been reported to interact with during the infection. Indeed, several viral proteins have been reported to interact with host cell proteins through their SH3 domains. The first characterized viral SH3 domain host cell proteins through their SH3 domains. The first characterized viral SH3 domain ligand protein was the Nef protein of HIV-1 (Saksela et al., 1995). Nef is essential for HIV- ligand protein was the Nef protein of HIV-1 (Saksela et al., 1995). Nef is essential for HIV- 1 replication and plays an important role as a pathogenesis factor of HIV-1 infection. The 1 replication and plays an important role as a pathogenesis factor of HIV-1 infection. The best known cellular activities of Nef in host cell are dependent on its highly conserved best known cellular activities of Nef in host cell are dependent on its highly conserved SH3 binding motif, which has a consensus PxxPxR (Saksela, 2011). Disruption of the SH3 binding motif, which has a consensus PxxPxR (Saksela, 2011). Disruption of the polyproline motif in Nef results in impaired replication. In addition to previously polyproline motif in Nef results in impaired replication. In addition to previously mentioned Nef association with SH3 domains of Src-family kinases Hck and Fyn (Saksela mentioned Nef association with SH3 domains of Src-family kinases Hck and Fyn (Saksela et al., 1995), Nef has also been reported to interact with other family members, namely et al., 1995), Nef has also been reported to interact with other family members, namely c-Src, Lck and Lyn. The binding of Nef to Hck is one of the strongest reported interactions c-Src, Lck and Lyn. The binding of Nef to Hck is one of the strongest reported interactions between an SH3 domain and its ligand (Lee et al., 1995), whereas the binding of Nef to between an SH3 domain and its ligand (Lee et al., 1995), whereas the binding of Nef to other SFKs has a modest affinity. The interaction of Nef with Hck results in the activation other SFKs has a modest affinity. The interaction of Nef with Hck results in the activation of the kinase by disrupting the intramolecular interaction between the SH2-kinase linker of the kinase by disrupting the intramolecular interaction between the SH2-kinase linker and the SH3 domain (Moarefi et al., 1997). The intramolecular SH3-ligand interaction is and the SH3 domain (Moarefi et al., 1997). The intramolecular SH3-ligand interaction is a key mechanism in Src-family kinases. Trible et al. showed that among the Src-family a key mechanism in Src-family kinases. Trible et al. showed that among the Src-family kinases, Lyn and c-Src were also activated by the association of Nef with their SH3 kinases, Lyn and c-Src were also activated by the association of Nef with their SH3 domains (Trible et al., 2006). In contrast, they noticed that Fyn, Lck, Fgr and Yes were domains (Trible et al., 2006). In contrast, they noticed that Fyn, Lck, Fgr and Yes were

30 30 not activated by the interaction. As a matter of fact, the association of Nef with Lck and not activated by the interaction. As a matter of fact, the association of Nef with Lck and Fyn has been reported to lead to suppression of the kinase activity (Briggs et al., 2000; Fyn has been reported to lead to suppression of the kinase activity (Briggs et al., 2000; Greenway et al., 1996). Greenway et al., 1996). The NS5A protein of hepatitis C virus (HCV) is essential for both viral replication and The NS5A protein of hepatitis C virus (HCV) is essential for both viral replication and virion assembly (Ross-Thriepland and Harris, 2015). It contains one N-terminal and two virion assembly (Ross-Thriepland and Harris, 2015). It contains one N-terminal and two C-terminal polyproline motifs and is known to have several host cell interaction partners C-terminal polyproline motifs and is known to have several host cell interaction partners (Macdonald et al., 2004; Macdonald et al., 2005; Tan et al., 1999). No interaction (Macdonald et al., 2004; Macdonald et al., 2005; Tan et al., 1999). No interaction partners for the N-terminal class I PxxP motif has been found so far. Instead, the two partners for the N-terminal class I PxxP motif has been found so far. Instead, the two closely spaced class II polyproline motifs near the C-terminus of NS5A have been closely spaced class II polyproline motifs near the C-terminus of NS5A have been identified as a binding site for SH3 domains of numerous host cell proteins. The first C- identified as a binding site for SH3 domains of numerous host cell proteins. The first C- terminal PxxP binds to the SH3 domain of Src-family kinase Lyn, whereas the second C- terminal PxxP binds to the SH3 domain of Src-family kinase Lyn, whereas the second C- terminal PxxP has been reported to associate with the SH3 domains of other Src kinases, terminal PxxP has been reported to associate with the SH3 domains of other Src kinases, namely Hck, Lck and Fyn (Macdonald et al., 2004) as well as with the adaptor protein namely Hck, Lck and Fyn (Macdonald et al., 2004) as well as with the adaptor protein Grb2 (growth factor receptor-bouns protein 2) (Tan et al., 1999). The interaction of NS5A Grb2 (growth factor receptor-bouns protein 2) (Tan et al., 1999). The interaction of NS5A with the SH3 domains of the Src-family kinases results in differential regulation of the with the SH3 domains of the Src-family kinases results in differential regulation of the kinase activity. While the activity of Hck, Lck and Lyn is suppressed by the interaction, kinase activity. While the activity of Hck, Lck and Lyn is suppressed by the interaction, the activity of Fyn is stimulated (Macdonald et al., 2004). The stimulation of Fyn by NS5A the activity of Fyn is stimulated (Macdonald et al., 2004). The stimulation of Fyn by NS5A binding to its SH3 domain was linked to tyrosine phosphorylation of transcription factor binding to its SH3 domain was linked to tyrosine phosphorylation of transcription factor Stat3. Since the constitutive phosphorylation of Stat3 is associated with many tumours, Stat3. Since the constitutive phosphorylation of Stat3 is associated with many tumours, the Fyn mediated phosphorylation of Stat3 was hypothesized to be involved in the the Fyn mediated phosphorylation of Stat3 was hypothesized to be involved in the development of hepatocellular carcinoma in HCV-infection. The interaction of NS5A development of hepatocellular carcinoma in HCV-infection. The interaction of NS5A with the SH3 domain of Grb2 was reported to disturb vaccinia virus induced with the SH3 domain of Grb2 was reported to disturb vaccinia virus induced phosphorylation of ERK1/2 (extracellular signal-regulated kinases 1 and 2) (Tan et al., phosphorylation of ERK1/2 (extracellular signal-regulated kinases 1 and 2) (Tan et al., 1999). The second C-terminal SH3 binding motif also mediates the association of NS5A 1999). The second C-terminal SH3 binding motif also mediates the association of NS5A with the SH3 domains of amphiphysin II (Masumi et al., 2005), MLK3 (Mixed Lineage with the SH3 domains of amphiphysin II (Masumi et al., 2005), MLK3 (Mixed Lineage Kinase 3) (Amako et al., 2013) and an adaptor protein CMS (Cas ligand with multiple SH3 Kinase 3) (Amako et al., 2013) and an adaptor protein CMS (Cas ligand with multiple SH3 domains) (Igloi et al., 2015; Mankouri et al., 2009). Interaction with amphiphysin was domains) (Igloi et al., 2015; Mankouri et al., 2009). Interaction with amphiphysin was shown to decrease the phosphorylation of NS5A enabling efficient viral replication, since shown to decrease the phosphorylation of NS5A enabling efficient viral replication, since hyperphosphorylation of NS5A inhibits virus replication (Masumi et al., 2005). HCV hyperphosphorylation of NS5A inhibits virus replication (Masumi et al., 2005). HCV establishes a persistent infection, thus it is important for the virus to control apoptosis. establishes a persistent infection, thus it is important for the virus to control apoptosis. Indeed, the interaction of NS5A with the SH3 domain of MLK3, a serine threonine Indeed, the interaction of NS5A with the SH3 domain of MLK3, a serine threonine protein kinase that is a member of MAP3K (mitogen activated protein kinase kinase protein kinase that is a member of MAP3K (mitogen activated protein kinase kinase kinase) family, has been linked to inhibition of apoptosis. MLK3 activates p38MAPK kinase) family, has been linked to inhibition of apoptosis. MLK3 activates p38MAPK which leads to up-regulation of K+ channel (Kv2.1) and apoptosis (Amako et al., 2013; which leads to up-regulation of K+ channel (Kv2.1) and apoptosis (Amako et al., 2013; Mankouri et al., 2009). The interaction between the SH3 domain of MLK3 and the Mankouri et al., 2009). The interaction between the SH3 domain of MLK3 and the second C-terminal polyproline motif of NS5A was demonstrated to prevent this second C-terminal polyproline motif of NS5A was demonstrated to prevent this activation of Kv2.1 and oxidative-stress induced apoptosis by disturbing activation of Kv2.1 and oxidative-stress induced apoptosis by disturbing MLK3/p38MAPK signaling (Amako et al., 2013). Finally, association of NS5A with the SH3 MLK3/p38MAPK signaling (Amako et al., 2013). Finally, association of NS5A with the SH3 domain of CMS disrupts the EGFR (epidermal growth factor receptor) trafficking and domain of CMS disrupts the EGFR (epidermal growth factor receptor) trafficking and ubiquitination (Igloi et al., 2015). EGFR is a key factor for HCV entry and replication (Diao ubiquitination (Igloi et al., 2015). EGFR is a key factor for HCV entry and replication (Diao et al., 2012). et al., 2012).

1.6 Crk adaptor proteins 1.6 Crk adaptor proteins The history of Crk proteins starts in 1988 when Mayer et al. isolated the v-crk (or gag- The history of Crk proteins starts in 1988 when Mayer et al. isolated the v-crk (or gag- crk) oncogene from chicken tumor virus number 10 (CT10) retrovirus (Mayer et al., crk) oncogene from chicken tumor virus number 10 (CT10) retrovirus (Mayer et al., 1988). It was named CT10 regulator of kinase (Crk) for its proposed function, since it was 1988). It was named CT10 regulator of kinase (Crk) for its proposed function, since it was able to increase cellular tyrosine phosphorylation and transform primary chicken able to increase cellular tyrosine phosphorylation and transform primary chicken

31 31 embryo fibroblasts but was lacking any catalytic domain. v-crk is a fusion gene of the embryo fibroblasts but was lacking any catalytic domain. v-crk is a fusion gene of the viral gag gene and a cellular gene which encodes two separate domains (see figure 6). viral gag gene and a cellular gene which encodes two separate domains (see figure 6). Couple of years after the finding of v-Crk, Reichman et al. isolated the cellular homolog Couple of years after the finding of v-Crk, Reichman et al. isolated the cellular homolog of chicken Crk which had a similar structural organization to v-Crk but contained a ~50 of chicken Crk which had a similar structural organization to v-Crk but contained a ~50 aa proline rich linker and an additional C-terminal domain (Reichman et al., 1992). aa proline rich linker and an additional C-terminal domain (Reichman et al., 1992). Following the sequencing of cellular Crk in chickens, two isoforms of Crk (CrkI and CrkII) Following the sequencing of cellular Crk in chickens, two isoforms of Crk (CrkI and CrkII) and Crk-like (CrkL) were isolated (Matsuda et al., 1992), and the family of Crk adaptor and Crk-like (CrkL) were isolated (Matsuda et al., 1992), and the family of Crk adaptor proteins are considered to comprise these three members: CrkI, CrkII and CrkL. CrkI and proteins are considered to comprise these three members: CrkI, CrkII and CrkL. CrkI and Crk II are alternatively spliced products from one gene (Matsuda et al., 1992), whereas Crk II are alternatively spliced products from one gene (Matsuda et al., 1992), whereas CrkL is encoded by a distinct gene and shares high sequence homology with CrkII CrkL is encoded by a distinct gene and shares high sequence homology with CrkII (Galletta et al., 1999; ten Hoeve et al., 1993). (Galletta et al., 1999; ten Hoeve et al., 1993).

1.6.1 Stucture and binding specificities of Crk proteins 1.6.1 Stucture and binding specificities of Crk proteins The Crk proteins are relatively small adaptor proteins lacking any enzymatic activity The Crk proteins are relatively small adaptor proteins lacking any enzymatic activity (Feller, 2001). The predicted molecular masses for Crk family proteins are 28 kDa for (Feller, 2001). The predicted molecular masses for Crk family proteins are 28 kDa for CrkI, 40 kDa for CrkII, and 36 kDa for CrkL. They possess one N-terminal SH2 domain CrkI, 40 kDa for CrkII, and 36 kDa for CrkL. They possess one N-terminal SH2 domain followed by one (in CrkI) or two (in CrkII and CrkL) SH3 domains (see Figure 6). The SH3 followed by one (in CrkI) or two (in CrkII and CrkL) SH3 domains (see Figure 6). The SH3 domains are called the N-terminal (nSH3) and C-terminal (cSH3) according whether they domains are called the N-terminal (nSH3) and C-terminal (cSH3) according whether they are more N- or C-terminally located. The nSH3 and cSH3 domains are separated by an are more N- or C-terminally located. The nSH3 and cSH3 domains are separated by an approximately 50 residue long linker region. Compared to CrkL SH2, CrkI and CrkII SH2 approximately 50 residue long linker region. Compared to CrkL SH2, CrkI and CrkII SH2 domain possess an extra stretch of 17 aas that contain a proline-rich region (Anafi et al., domain possess an extra stretch of 17 aas that contain a proline-rich region (Anafi et al., 1996). The overall sequence identity of CrkII and CrkL is 56 %, and in the structural 1996). The overall sequence identity of CrkII and CrkL is 56 %, and in the structural domain regions 72 % (Kobashigawa and Inagaki, 2012). domain regions 72 % (Kobashigawa and Inagaki, 2012).

Figure 6. Schematic representation of the domain structure of the Crk adaptor proteins. Sequence Figure 6. Schematic representation of the domain structure of the Crk adaptor proteins. Sequence identities are shown for CrkII and CrkL. After Birge et al., 2009 and Kobashiwaga and Inagaki, 2012. identities are shown for CrkII and CrkL. After Birge et al., 2009 and Kobashiwaga and Inagaki, 2012.

The ligand binding specificities of the SH2 and nSH3 domains of CrkII and CrkL are similar. The ligand binding specificities of the SH2 and nSH3 domains of CrkII and CrkL are similar. For the SH2, the preferred binding motif is pYxxP (where pY means phosphorylated For the SH2, the preferred binding motif is pYxxP (where pY means phosphorylated tyrosine residue) (Songyang et al., 1993). Multiple pYxxP motifs are found in Crk SH2 tyrosine residue) (Songyang et al., 1993). Multiple pYxxP motifs are found in Crk SH2

32 32 binding proteins like (Birge et al., 1993), p130Cas (Sakai et al., 1994), and c-Cbl binding proteins like paxillin (Birge et al., 1993), p130Cas (Sakai et al., 1994), and c-Cbl (Ribon et al., 1996). CrkII SH2 domain has also been reported to bind a pYxxL motif in (Ribon et al., 1996). CrkII SH2 domain has also been reported to bind a pYxxL motif in the cell cycle regulator Wee1 (Smith et al., 2000). In general, the nSH3 of Crk proteins the cell cycle regulator Wee1 (Smith et al., 2000). In general, the nSH3 of Crk proteins bind to class II polyproline motifs, and the consensus binding motif is PxxPxK (Knudsen bind to class II polyproline motifs, and the consensus binding motif is PxxPxK (Knudsen et al., 1994; Tanaka et al., 1994). The lysine (K) residue as a positive residue in the et al., 1994; Tanaka et al., 1994). The lysine (K) residue as a positive residue in the consensus was found to be critical for high affinity binding of C3G to the nSH3 domain consensus was found to be critical for high affinity binding of C3G to the nSH3 domain of Crk proteins (Knudsen et al., 1995). Changing the lysine to arginine, which normally is of Crk proteins (Knudsen et al., 1995). Changing the lysine to arginine, which normally is the preferred positive residue in SH3 binding motifs, strongly reduced the affinity of the preferred positive residue in SH3 binding motifs, strongly reduced the affinity of C3G-derived peptide for the nSH3 domain. The remarkable affinity and selectivity C3G-derived peptide for the nSH3 domain. The remarkable affinity and selectivity created by the lysine residue over arginine was explained by the unique structure of the created by the lysine residue over arginine was explained by the unique structure of the nSH3 domain specificity-pocket, where three acidic residues is involved in forming nSH3 domain specificity-pocket, where three acidic residues is involved in forming hydrogen bonds with lysine (Wu et al., 1995). In contrast, the arginine side chain is not hydrogen bonds with lysine (Wu et al., 1995). In contrast, the arginine side chain is not well ordered and does not adopt an optimal conformation for hydrogen bonds. The cSH3 well ordered and does not adopt an optimal conformation for hydrogen bonds. The cSH3 domain is an atypical SH3 domain, since the conserved residues in the hydrophobic domain is an atypical SH3 domain, since the conserved residues in the hydrophobic polyproline binding pockets are replaced by polar residues, and so far no polyproline polyproline binding pockets are replaced by polar residues, and so far no polyproline containing binding partners have been identified for the cSH3 (Muralidharan et al., containing binding partners have been identified for the cSH3 (Muralidharan et al., 2006). Instead, cSH3 is involved in regulation and nuclear export of the Crk proteins (see 2006). Instead, cSH3 is involved in regulation and nuclear export of the Crk proteins (see below). below).

1.6.2 Regulation of Crk proteins 1.6.2 Regulation of Crk proteins The linker region between nSH3 and cSH3 domains of CrkII and CrkL contains a tyrosine The linker region between nSH3 and cSH3 domains of CrkII and CrkL contains a tyrosine residue (Y221 in CrkII and Y207 in CrkL; Figure 6) that is important for the regulation of residue (Y221 in CrkII and Y207 in CrkL; Figure 6) that is important for the regulation of these proteins. When phosphorylated by tyrosine kinase c-Abl, the tyrosine in the linker these proteins. When phosphorylated by tyrosine kinase c-Abl, the tyrosine in the linker region of CrkII and CrkL binds to its own SH2 domain to form a closed structure that region of CrkII and CrkL binds to its own SH2 domain to form a closed structure that prevents the SH2 domain from binding to its tyrosine phosphorylated ligand proteins prevents the SH2 domain from binding to its tyrosine phosphorylated ligand proteins (de Jong et al., 1997; Feller et al., 1994; Kobashigawa et al., 2007; Rosen et al., 1995). In (de Jong et al., 1997; Feller et al., 1994; Kobashigawa et al., 2007; Rosen et al., 1995). In addition, the closed structure prevents the binding of nSH3 domain of CrkII ligands, but addition, the closed structure prevents the binding of nSH3 domain of CrkII ligands, but in CrkL the nSH3 domain is fully accessible (Jankowski et al., 2012). in CrkL the nSH3 domain is fully accessible (Jankowski et al., 2012). NMR studies with chicken CrkII revealed another auto-inhibition regulation method by NMR studies with chicken CrkII revealed another auto-inhibition regulation method by a cis-trans isomerization at a glycine-proline (G237-P238) peptide bond located at the a cis-trans isomerization at a glycine-proline (G237-P238) peptide bond located at the cSH3 boundary (see figure 7) (Sarkar et al., 2007; Sarkar et al., 2011). In the cis cSH3 boundary (see figure 7) (Sarkar et al., 2007; Sarkar et al., 2011). In the cis conformer, nSH3 and cSH3 are able to interact with each other when three residues of conformer, nSH3 and cSH3 are able to interact with each other when three residues of cSH3 (P238, F239 and I270) occupy the PPII binding site on nSH3 in a manner similar to cSH3 (P238, F239 and I270) occupy the PPII binding site on nSH3 in a manner similar to a PPII peptide. This prevents the binding of nSH3 domain to its natural ligands. In a PPII peptide. This prevents the binding of nSH3 domain to its natural ligands. In contrast, the trans conformer adopts an open state where the PPII binding site on nSH3 contrast, the trans conformer adopts an open state where the PPII binding site on nSH3 is able to bind PPII ligands. In human CrkII, residues 224-237 (called the inter SH3 core; is able to bind PPII ligands. In human CrkII, residues 224-237 (called the inter SH3 core; ISC) within the cSH3 form contact the SH2 and both SH3 domains (Kobashigawa et al., ISC) within the cSH3 form contact the SH2 and both SH3 domains (Kobashigawa et al., 2007). These contacts arrange the three domains into a compact structure and represses 2007). These contacts arrange the three domains into a compact structure and represses the interaction of nSH3 domain with its ligands. the interaction of nSH3 domain with its ligands.

33 33 Figure 7. Schematic representation of cis-trans isomerization in Crk II. After Sarkar et al., 2007. Figure 7. Schematic representation of cis-trans isomerization in Crk II. After Sarkar et al., 2007.

The solution NMR structure of CrkL revealed that the overall domain organization in CrkL The solution NMR structure of CrkL revealed that the overall domain organization in CrkL is very different from CrkII (Jankowski et al., 2012). The binding site of SH2 domain is is very different from CrkII (Jankowski et al., 2012). The binding site of SH2 domain is partially masked in CrkL, while in CrkII it is fully accessible. In addition, the cSH3 of CrkL partially masked in CrkL, while in CrkII it is fully accessible. In addition, the cSH3 of CrkL does not seem to have similar auto-inhibition activity as in CrkII. In CrkL the cSH3 domain does not seem to have similar auto-inhibition activity as in CrkII. In CrkL the cSH3 domain is mobile and does not interact with any of the other domains. Thus, the binding site of is mobile and does not interact with any of the other domains. Thus, the binding site of nSH3 domain is fully accessible in CrkL, while in CrkII it is masked by the ISC. nSH3 domain is fully accessible in CrkL, while in CrkII it is masked by the ISC. Finally, the cSH3 domain has been shown to possess a positive regulatory role as well. Finally, the cSH3 domain has been shown to possess a positive regulatory role as well. In contrast to conventional SH3 domains, cSH3 has a conserved PNAY-motif in the RT- In contrast to conventional SH3 domains, cSH3 has a conserved PNAY-motif in the RT- loop (Reichman et al., 2005). The tyrosine residue, Y251, within the PNAY-motif was loop (Reichman et al., 2005). The tyrosine residue, Y251, within the PNAY-motif was shown to be phosphorylated by c-Abl (Reichman et al., 2005; Sriram et al., 2011). shown to be phosphorylated by c-Abl (Reichman et al., 2005; Sriram et al., 2011). Phosphorylation at Y251 enables the association of the c-Abl SH2 domain with this site, Phosphorylation at Y251 enables the association of the c-Abl SH2 domain with this site, which enhances the activation of Abl. In contrast, Jankowski et al. reported that in CrkL which enhances the activation of Abl. In contrast, Jankowski et al. reported that in CrkL only one tyrosine residue is phosphorylated by c-Abl in vitro (Jankowski et al., 2012). only one tyrosine residue is phosphorylated by c-Abl in vitro (Jankowski et al., 2012).

1.6.3 Nuclear import and export of Crk proteins 1.6.3 Nuclear import and export of Crk proteins Crk proteins are predominantly found in the cytoplasm, but they also are able to enter Crk proteins are predominantly found in the cytoplasm, but they also are able to enter the nucleus, although they do not seem to have an NLS. Therefore, they are thought to the nucleus, although they do not seem to have an NLS. Therefore, they are thought to be imported to the nucleus through their interaction with other proteins that contain be imported to the nucleus through their interaction with other proteins that contain an NLS (Kar et al., 2007). The interaction of nSH3 with Wee1, and c-Abl has an NLS (Kar et al., 2007). The interaction of nSH3 with Wee1, DOCK180 and c-Abl has been reported to mediate the nuclear translocation of CrkII (Kar et al., 2007). For been reported to mediate the nuclear translocation of CrkII (Kar et al., 2007). For example, in CrkII-Wee1 complex, the NES of CrkII is masked, retaining CrkII in the example, in CrkII-Wee1 complex, the NES of CrkII is masked, retaining CrkII in the nucleus. On the contrary, the nuclear export of Crk proteins is an active process, which nucleus. On the contrary, the nuclear export of Crk proteins is an active process, which is mediated by the interaction of NES, located in cSH3 domain, with a nuclear export is mediated by the interaction of NES, located in cSH3 domain, with a nuclear export receptor Crm1/exportin (chromosome maintenance region-1) (Smith et al., 2002). receptor Crm1/exportin (chromosome maintenance region-1) (Smith et al., 2002). Harkiolaki et al. reported that CrkL cSH3 can exist in monomeric or dimeric conformation Harkiolaki et al. reported that CrkL cSH3 can exist in monomeric or dimeric conformation (Harkiolaki et al., 2006). The crystal structure of the cSH3 revealed that the NES is mostly (Harkiolaki et al., 2006). The crystal structure of the cSH3 revealed that the NES is mostly buried under the domain surface in both conformations. However, upon buried under the domain surface in both conformations. However, upon dimer/monomer transition, partial unfolding of the cSH3 exposes the NES and the dimer/monomer transition, partial unfolding of the cSH3 exposes the NES and the complex with Crm1/exportin can form. complex with Crm1/exportin can form.

1.6.4 Biological function of Crk proteins 1.6.4 Biological function of Crk proteins The members of the Crk family are involved in several signal transduction pathways The members of the Crk family are involved in several signal transduction pathways downstream of a wide range of receptors (Birge et al., 2009; Feller, 2001). Like other downstream of a wide range of receptors (Birge et al., 2009; Feller, 2001). Like other

34 34 adaptor proteins, they physically bridge tyrosine phosphorylated proteins to various adaptor proteins, they physically bridge tyrosine phosphorylated proteins to various intracellular signaling pathways. The SH2 domain serves as an input pathway by binding intracellular signaling pathways. The SH2 domain serves as an input pathway by binding to activated receptors and the signal is then transmitted through the interaction to activated receptors and the signal is then transmitted through the interaction partners of nSH3 domain. Crk proteins have been reported to associate with numerous partners of nSH3 domain. Crk proteins have been reported to associate with numerous cellular proteins through their SH2 and SH3 domains and they have important regulatory cellular proteins through their SH2 and SH3 domains and they have important regulatory roles in numerous cellular signaling processes, including cell adhesion, growth, roles in numerous cellular signaling processes, including cell adhesion, growth, differentiation, proliferation, transformation, and apoptosis. differentiation, proliferation, transformation, and apoptosis. Although Crk proteins are ubiquitously expressed, and their SH2 and SH3 domains are Although Crk proteins are ubiquitously expressed, and their SH2 and SH3 domains are highly homologous with similar binding preferences, they seem to have distinct, non- highly homologous with similar binding preferences, they seem to have distinct, non- overlapping roles during embryonic development. Knockout of CrkI/II or CrkL leads to overlapping roles during embryonic development. Knockout of CrkI/II or CrkL leads to different developmental defects and the mice die perinatally (Guris et al., 2001; Park et different developmental defects and the mice die perinatally (Guris et al., 2001; Park et al., 2006). Mice lacking the CrkI and CrkII die perinatally due to defects in cardiovascular al., 2006). Mice lacking the CrkI and CrkII die perinatally due to defects in cardiovascular and craniofacial development (Park et al., 2006), whereas the CrkL null mice have and craniofacial development (Park et al., 2006), whereas the CrkL null mice have defects in neural crest and cardiac development (Guris et al., 2001). Furthermore, in defects in neural crest and cardiac development (Guris et al., 2001). Furthermore, in mouse embryonic fibroblasts (MEFs) where CrkII or CrkL has been depleted the other mouse embryonic fibroblasts (MEFs) where CrkII or CrkL has been depleted the other protein does not compensate the loss of the other by overexpression, again suggesting protein does not compensate the loss of the other by overexpression, again suggesting that each of the two genes has essential biological functions that cannot be replaced by that each of the two genes has essential biological functions that cannot be replaced by the other gene. Mice that lack CrkII but still expresses CrkI develop normally, suggesting the other gene. Mice that lack CrkII but still expresses CrkI develop normally, suggesting that CrkI can compensate the loss of CrkII in development, but they die within a few days that CrkI can compensate the loss of CrkII in development, but they die within a few days after birth by unknown mechanisms (Imaizumi et al., 1999). after birth by unknown mechanisms (Imaizumi et al., 1999). However, Crk proteins have been shown to have essential overlapping roles as well. However, Crk proteins have been shown to have essential overlapping roles as well. Tissue-specific contribution of Crk proteins has been studied by generating floxed alleles Tissue-specific contribution of Crk proteins has been studied by generating floxed alleles of Crk and CrkL. The lack of both CrkI/II and CrkL in skeletal muscle led to severe defects of Crk and CrkL. The lack of both CrkI/II and CrkL in skeletal muscle led to severe defects in the neuromuscular synapse, whereas mice lacking only one of the proteins did not in the neuromuscular synapse, whereas mice lacking only one of the proteins did not show any defects (Hallock et al., 2010). A recent study demonstrated that Crk proteins show any defects (Hallock et al., 2010). A recent study demonstrated that Crk proteins play overlapping roles in maintaining cell structure and motility in MEF cells (Park and play overlapping roles in maintaining cell structure and motility in MEF cells (Park and Curran, 2014). The lack of both genes resulted in smaller cell size, while the lack of only Curran, 2014). The lack of both genes resulted in smaller cell size, while the lack of only Crk or CrkL did not have any effect on cell morphology. Crk or CrkL did not have any effect on cell morphology.

1.6.4.1 Crk in PI3K signaling 1.6.4.1 Crk in PI3K signaling Crk proteins have been reported to participate in the regulation of PI3K signaling. The Crk proteins have been reported to participate in the regulation of PI3K signaling. The viral oncoprotein v-Crk activate PI3K/Akt pathway in CEF (chicken embryonic fibroblast) viral oncoprotein v-Crk activate PI3K/Akt pathway in CEF (chicken embryonic fibroblast) cells (Akagi et al., 2000). Both SH2 and SH3 domains are important for the activation, cells (Akagi et al., 2000). Both SH2 and SH3 domains are important for the activation, since SH2- or SH3-mutant forms of v-Crk were not able to activate the pathway. The v- since SH2- or SH3-mutant forms of v-Crk were not able to activate the pathway. The v- Crk SH2 domain was demonstrated to be involved in PI3K activation by mediating the Crk SH2 domain was demonstrated to be involved in PI3K activation by mediating the activation of Src family kinases which in turn phosphorylate focal adhesion kinase (FAK) activation of Src family kinases which in turn phosphorylate focal adhesion kinase (FAK) (Akagi et al., 2002). Tyrosine phosphorylated FAK then associates with the SH2 domain (Akagi et al., 2002). Tyrosine phosphorylated FAK then associates with the SH2 domain of p85 regulatory subunit of PI3K, which was shown to be essential for the pathway of p85 regulatory subunit of PI3K, which was shown to be essential for the pathway activation. The v-Crk SH3 domain was demonstrated to participate in the induction of activation. The v-Crk SH3 domain was demonstrated to participate in the induction of the pathway by activating H-Ras, a member of Ras family of small G proteins, which in the pathway by activating H-Ras, a member of Ras family of small G proteins, which in turn binds to the p110 catalytic subunit of PI3K and may activate it. The activation of H- turn binds to the p110 catalytic subunit of PI3K and may activate it. The activation of H- Ras was shown to be mediated by binding of SOS (son of sevenless), a guanine Ras was shown to be mediated by binding of SOS (son of sevenless), a guanine nucleotide exchange factor, to the SH3 domain of v-Crk. nucleotide exchange factor, to the SH3 domain of v-Crk. In addition, Crk proteins have been reported to directly interact with the p85 regulatory In addition, Crk proteins have been reported to directly interact with the p85 regulatory subunit of PI3K through the Crk nSH3 domain and a proline-rich motif in p85 (Gelkop et subunit of PI3K through the Crk nSH3 domain and a proline-rich motif in p85 (Gelkop et al., 2001; Sattler et al., 1997). The interaction was shown to be involved in PI3K al., 2001; Sattler et al., 1997). The interaction was shown to be involved in PI3K

35 35 regulation upon immune cell activation, where a trimeric complex of Cbl, a ubiquitin regulation upon immune cell activation, where a trimeric complex of Cbl, a ubiquitin ligase, CrkII and p85 is formed (Gelkop et al., 2001). The Crk-p85-Cbl complex has also ligase, CrkII and p85 is formed (Gelkop et al., 2001). The Crk-p85-Cbl complex has also been shown to be involved in oncogenic signaling in chronic myeloid leukemia (Sattler been shown to be involved in oncogenic signaling in chronic myeloid leukemia (Sattler et al., 1996). Binding of CrkL SH3 domain to Bcr-Abl fusion protein resulted in et al., 1996). Binding of CrkL SH3 domain to Bcr-Abl fusion protein resulted in phosphorylation of CrkL and subsequent binding to the SH2 domain of Cbl. Cbl is then phosphorylation of CrkL and subsequent binding to the SH2 domain of Cbl. Cbl is then phosphorylated which leads to its association with p85. phosphorylated which leads to its association with p85.

1.6.4.2 Crk in apoptotic pathways 1.6.4.2 Crk in apoptotic pathways CrkII was shown to be required for apoptosis in Xenopus egg extract by activating CrkII was shown to be required for apoptosis in Xenopus egg extract by activating caspases (Evans et al., 1997). Depletion of CrkII from egg extracts prevented apoptosis, caspases (Evans et al., 1997). Depletion of CrkII from egg extracts prevented apoptosis, and further studies indicated that the cell cycle regulatory protein Wee1 interacts with and further studies indicated that the cell cycle regulatory protein Wee1 interacts with CrkII SH2 domain (Smith et al., 2000). The involvement of CrkII-Wee1 interaction in CrkII SH2 domain (Smith et al., 2000). The involvement of CrkII-Wee1 interaction in apoptosis was further studied in mammalian cells (Smith et al., 2002). This study apoptosis was further studied in mammalian cells (Smith et al., 2002). This study demonstrated an NES sequence found in the cSH3 domain of CrkII. It was found to demonstrated an NES sequence found in the cSH3 domain of CrkII. It was found to mediate the binding of CrkII to Crm1 and subsequent nuclear export of CrkII. Mutation mediate the binding of CrkII to Crm1 and subsequent nuclear export of CrkII. Mutation of this sequence enhanced the association of CrkII with Wee1. Furthermore, the of this sequence enhanced the association of CrkII with Wee1. Furthermore, the proapoptotic activity of CrkII was increased when the NES was impaired, further proapoptotic activity of CrkII was increased when the NES was impaired, further implying that the proapoptotic function of CrkII depends on nuclear localization. Indeed, implying that the proapoptotic function of CrkII depends on nuclear localization. Indeed, specific targeting of CrkII by adding an NLS was shown to spontaneously activate specific targeting of CrkII by adding an NLS was shown to spontaneously activate apoptosis (Kar et al., 2007). apoptosis (Kar et al., 2007). CrkI and CrkII have also been shown to be required for ER (endoplasmic reticulum) CrkI and CrkII have also been shown to be required for ER (endoplasmic reticulum) stress-induced apoptosis, which is mediated by the mitochondrial apoptotic pathway stress-induced apoptosis, which is mediated by the mitochondrial apoptotic pathway (Austgen et al., 2012). CrkI/CrkII KO MEFs were resistant to ER stress-induced apoptosis. (Austgen et al., 2012). CrkI/CrkII KO MEFs were resistant to ER stress-induced apoptosis. In addition, ER stress was found to induce proteolytic cleavage of CrkI/II by a cysteine In addition, ER stress was found to induce proteolytic cleavage of CrkI/II by a cysteine protease. This about 14 kDa N-terminal fragment was further shown to have enhanced protease. This about 14 kDa N-terminal fragment was further shown to have enhanced apoptotic activity when transiently expressed. The proapoptotic function of the apoptotic activity when transiently expressed. The proapoptotic function of the fragment was found to be due to CrkI/II binding to an antiapoptotic Bcl-2 family protein, fragment was found to be due to CrkI/II binding to an antiapoptotic Bcl-2 family protein, Bcl-XL, upon ER stress induction. The interaction of CrkI/II with Bcl-XL is mediated by a Bcl-XL, upon ER stress induction. The interaction of CrkI/II with Bcl-XL is mediated by a BH3 (Bcl2 homology 3) found in the 14 kDa N-terminal fragment. BH3 (Bcl2 homology 3) death domain found in the 14 kDa N-terminal fragment. Approximately 9-12 amino acid long BH3 domain is involved in activation or inhibition Approximately 9-12 amino acid long BH3 domain is involved in activation or inhibition of proapoptotic or antiapoptotic proteins, respectively (Lomonosova and Chinnadurai, of proapoptotic or antiapoptotic proteins, respectively (Lomonosova and Chinnadurai, 2008). 2008).

36 36 2 AIMS OF THE STUDY 2 AIMS OF THE STUDY

The aim of this doctoral thesis was to study influenza A virus NS1 protein and its host The aim of this doctoral thesis was to study influenza A virus NS1 protein and its host cell SH3 domain interactions. More specifically the aims were: cell SH3 domain interactions. More specifically the aims were:

1. to characterize a putative SH3 binding motif in IAV NS1 protein, its host cell SH3- 1. to characterize a putative SH3 binding motif in IAV NS1 protein, its host cell SH3- domain interaction partners, and the functional role of these interactions. domain interaction partners, and the functional role of these interactions. 2. to study the role and molecular mechanism of Crk-NS1 interaction for the 2. to study the role and molecular mechanism of Crk-NS1 interaction for the enhanced PI3K activation by NS1 protein. enhanced PI3K activation by NS1 protein. 3. to study the subcellular localization of the Crk-NS1 complex and its role in the 3. to study the subcellular localization of the Crk-NS1 complex and its role in the host cell. host cell.

37 37 3 MATERIALS AND METHODS 3 MATERIALS AND METHODS

3.1 Cell culture and transfections (I-III) 3.1 Cell culture and transfections (I-III) Human embryonic kidney fibroblast (293T) cell line (I,II) was purchased from ATCC, Human embryonic kidney fibroblast (293T) cell line (I,II) was purchased from ATCC, human hepatocellular carcinoma (Huh7) cell line (I-III) was obtained from Mark Harris human hepatocellular carcinoma (Huh7) cell line (I-III) was obtained from Mark Harris (University of Leeds, England) , and human lung adenocarcinoma epithelial (A549) cell (University of Leeds, England) , and human lung adenocarcinoma epithelial (A549) cell line (I,III) was received from Ilkka Julkunen (THL, Helsinki, Finland). The cells were line (I,III) was received from Ilkka Julkunen (THL, Helsinki, Finland). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Aldrich) supplemented cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Aldrich) supplemented with 4500 mg/L of glucose, 10% fetal bovine serum (FBS) (Gibco), 0.05 mg/ml penicillin, with 4500 mg/L of glucose, 10% fetal bovine serum (FBS) (Gibco), 0.05 mg/ml penicillin, 0.05 mg/ml streptomycin (Sigma Aldrich), and 1 mM L-glutamine (Sigma Aldrich) and 0.05 mg/ml streptomycin (Sigma Aldrich), and 1 mM L-glutamine (Sigma Aldrich) and were maintained in a humidified atmosphere in the presence of 5% CO2 at 37 °C. were maintained in a humidified atmosphere in the presence of 5% CO2 at 37 °C. Transient transfection of 293T cells was performed by the calcium phosphate Transient transfection of 293T cells was performed by the calcium phosphate precipitation method. Briefly: 10-20 μg of plasmid DNA was mixed with 250 μl of water, precipitation method. Briefly: 10-20 μg of plasmid DNA was mixed with 250 μl of water, and 250 μl of 2.5 M CaCl2 was added and mixed. The plasmid mix was added dropwise and 250 μl of 2.5 M CaCl2 was added and mixed. The plasmid mix was added dropwise in 500 μl of 2xHeBS (274 mM NaCl, 10 mM KCl, 1,4 mM Na2HPO4, 15 mM glucose, 42 in 500 μl of 2xHeBS (274 mM NaCl, 10 mM KCl, 1,4 mM Na2HPO4, 15 mM glucose, 42 mM Hepes, pH 7.05) while vortexing. The transfection mix was kept in RT for 20-30 mM Hepes, pH 7.05) while vortexing. The transfection mix was kept in RT for 20-30 minutes and added dropwise on top of the cells in 10 cm culture plate and the medium minutes and added dropwise on top of the cells in 10 cm culture plate and the medium was changed after 12 hours. Huh7 and A549 cells were transfected with was changed after 12 hours. Huh7 and A549 cells were transfected with Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions.

3.2 Plasmid constructs 3.2 Plasmid constructs

3.2.1 Eukaryotic expression vectors (I-III) 3.2.1 Eukaryotic expression vectors (I-III) The cDNA fragment for A/Mallard NS1 protein was cloned from the total cellular RNA of The cDNA fragment for A/Mallard NS1 protein was cloned from the total cellular RNA of cells infected with A/mallard/Netherlands/12/2000 (H7N3) IAV strain by standard cells infected with A/mallard/Netherlands/12/2000 (H7N3) IAV strain by standard methods. Synthetic cDNA encoding A/Brevig Mission/1/18 (H1N1) IAV strain, so called methods. Synthetic cDNA encoding A/Brevig Mission/1/18 (H1N1) IAV strain, so called the Spanish Flu, NS1 protein (A/Brevig) was purchased from GENEART. The cDNAs for the Spanish Flu, NS1 protein (A/Brevig) was purchased from GENEART. The cDNAs for NS1 proteins of A/WSN/33 (H1N1) (A/WSN) and A/Udorn/72 (H3N2) (A/Udorn) IAVs NS1 proteins of A/WSN/33 (H1N1) (A/WSN) and A/Udorn/72 (H3N2) (A/Udorn) IAVs were obtained from Ilkka Julkunen (THL, Helsinki, Finland). The cDNAs for all NS1 were obtained from Ilkka Julkunen (THL, Helsinki, Finland). The cDNAs for all NS1 proteins were cloned into the pEBB vector (Tanaka et al., 1995) containing an N-terminal proteins were cloned into the pEBB vector (Tanaka et al., 1995) containing an N-terminal myc epitope tag. Codon changes in NS1 gene were generated by standard overlap PCR myc epitope tag. Codon changes in NS1 gene were generated by standard overlap PCR mutagenesis. In NS1-Cyto, the NLS1 was mutated (R38A, K41A) and a NES from MAPKK1 mutagenesis. In NS1-Cyto, the NLS1 was mutated (R38A, K41A) and a NES from MAPKK1 (LQKKLEELEL) was inserted before the NS1 coding sequence. The cDNA for CrkI, CrkII and (LQKKLEELEL) was inserted before the NS1 coding sequence. The cDNA for CrkI, CrkII and CrkL was cloned into the pEBB vector with a C-terminal biotin acceptor domain (PP- CrkL was cloned into the pEBB vector with a C-terminal biotin acceptor domain (PP- domain) from PinPoint-Xa1 T-vector (Promega). The full-length cDNA for p85β was domain) from PinPoint-Xa1 T-vector (Promega). The full-length cDNA for p85β was purchased from the Genome Biology Unit (MGC-collection, University of Helsinki) and purchased from the Genome Biology Unit (MGC-collection, University of Helsinki) and cloned in to the pEBB vector with an N-terminal HA epitope tag or PP-domain. The codon cloned in to the pEBB vector with an N-terminal HA epitope tag or PP-domain. The codon changes to p85β sequence were done by overlap PCR mutagenesis. The NS1 and p85β changes to p85β sequence were done by overlap PCR mutagenesis. The NS1 and p85β mutations used in this study are summarized in Table 2. mutations used in this study are summarized in Table 2. Fluorescent fusion proteins were generated by fusing a red fluorescent protein, Fluorescent fusion proteins were generated by fusing a red fluorescent protein, mCherry, to the N-terminus of A/Mallard NS1 and A/Mallard NS1-Cyto, and a green mCherry, to the N-terminus of A/Mallard NS1 and A/Mallard NS1-Cyto, and a green fluorescent protein, eGFP, to the N-terminus of CrkII and CrkL. The ISRE-Luc reporter fluorescent protein, eGFP, to the N-terminus of CrkII and CrkL. The ISRE-Luc reporter plasmid was obtained from James E. Darnell Jr. (Rockefeller University, New York) and plasmid was obtained from James E. Darnell Jr. (Rockefeller University, New York) and contains an interferon-stimulated response element (ISRE) -containing fragment in front contains an interferon-stimulated response element (ISRE) -containing fragment in front of a minimal thymidine kinase promoter driving firefly luciferase expression. Renilla of a minimal thymidine kinase promoter driving firefly luciferase expression. Renilla

38 38 luciferase cDNA from pRL-null vector (Promega) was cloned into pcDNA3.1 vector to luciferase cDNA from pRL-null vector (Promega) was cloned into pcDNA3.1 vector to create pcDNA-Renilla. create pcDNA-Renilla.

Table 2. NS1 and p85β mutants used in this study. Table 2. NS1 and p85β mutants used in this study.

Mutation Acronym Functional consequence Study Mutation Acronym Functional consequence Study

A/Mallard or A/Brevig NS1 A/Mallard or A/Brevig NS1

P212A,P215A AxxA/M1 Disrupts the SH3 binding consensus I,II P212A,P215A AxxA/M1 Disrupts the SH3 binding consensus I,II

P213A, P216A M2 No effect I P213A, P216A M2 No effect I

K217E M3 Disrupts the SH3 binding consensus I K217E M3 Disrupts the SH3 binding consensus I

K217R M4 Alters the specificity of SH3 binding site I K217R M4 Alters the specificity of SH3 binding site I

P215T M5 Disrupts the SH3 binding consensus, I P215T M5 Disrupts the SH3 binding consensus, I

mimics the human IAV NS1 sequence I mimics the human IAV NS1 sequence I

Y89F Prevents binding to p85β II Y89F Prevents binding to p85β II

R38A, K41A + additional NES NS1-Cyto Prevents nuclear localization III R38A, K41A + additional NES NS1-Cyto Prevents nuclear localization III

p85β p85β

V573M Prevents binding to NS1 II V573M Prevents binding to NS1 II

P294A,P297A Prevents binding to Crk nSH3 domain II P294A,P297A Prevents binding to Crk nSH3 domain II

3.2.2 Bacterial expression vectors (I,II) 3.2.2 Bacterial expression vectors (I,II) The GST (Gluthathione S-transferase) and MBP (maltose binding protein) –NS1 The GST (Gluthathione S-transferase) and MBP (maltose binding protein) –NS1 constructs were generated by cloning the cDNAs of A/Mallard and A/Brevig into pGEX- constructs were generated by cloning the cDNAs of A/Mallard and A/Brevig into pGEX- 4T-1 vector (GE Healthcare) and pMAL-c2x (New England Biolabs), respectively. GST-PP- 4T-1 vector (GE Healthcare) and pMAL-c2x (New England Biolabs), respectively. GST-PP- SH3 constructs for CrkII, CrkL, p85α, p85β, and Eps8L1 SH3 domains were generated by SH3 constructs for CrkII, CrkL, p85α, p85β, and Eps8L1 SH3 domains were generated by transferring the codon optimized cDNAs from the human SH3 domain library transferring the codon optimized cDNAs from the human SH3 domain library (Kärkkäinen et al., 2006) into pGEX-4T-1 which had a PP-domain inserted between the (Kärkkäinen et al., 2006) into pGEX-4T-1 which had a PP-domain inserted between the GST and multiple cloning site. Coding sequence for the peptide CLNCFRPLPPLPPPPR was GST and multiple cloning site. Coding sequence for the peptide CLNCFRPLPPLPPPPR was cloned into the pMAL-c2x vector to generate the construct for p85α-BP (p85α binding cloned into the pMAL-c2x vector to generate the construct for p85α-BP (p85α binding peptide). peptide).

3.3 Viruses and virus infections (I,III) 3.3 Viruses and virus infections (I,III) The wt IAV strains used in this study were: A/mallard/Netherlands/12/2000 (H7N3), The wt IAV strains used in this study were: A/mallard/Netherlands/12/2000 (H7N3), A/Udorn/72 (H3N2), and A/WSN/1933 (H1N1). The recombinant IAVs were generated A/Udorn/72 (H3N2), and A/WSN/1933 (H1N1). The recombinant IAVs were generated as previously described (Neumann et al., 1999). The background virus for the as previously described (Neumann et al., 1999). The background virus for the recombinant IAV viruses used in this study was A/WSN/1933. The NS-segment recombinant IAV viruses used in this study was A/WSN/1933. The NS-segment originated either from A/WSN/1933 or A/mallard/Netherlands/12/2000 virus strains. originated either from A/WSN/1933 or A/mallard/Netherlands/12/2000 virus strains. The codon changes to the NS1 sequence in the NS-segment were done by overlapping The codon changes to the NS1 sequence in the NS-segment were done by overlapping

39 39 PCR mutagenesis. The recombinant IAVs and their mutations used in study III are PCR mutagenesis. The recombinant IAVs and their mutations used in study III are summarized in Table 3. summarized in Table 3. IAVs were propagated in 11-day-old embryonated chicken eggs at 34 °C for 3 days. Virus IAVs were propagated in 11-day-old embryonated chicken eggs at 34 °C for 3 days. Virus infections of A549 cells were carried out in normal medium supplemented with 2 % FBS infections of A549 cells were carried out in normal medium supplemented with 2 % FBS and in the presence of 5 μg/ml of TPCK-treated trypsin (Sigma Aldrich) for 20-24 hours and in the presence of 5 μg/ml of TPCK-treated trypsin (Sigma Aldrich) for 20-24 hours at a multiplicity of infection (MOI) of 0.5 to 5 depending on the assay. To metabolically at a multiplicity of infection (MOI) of 0.5 to 5 depending on the assay. To metabolically label virus-infected cells, the medium was changed 4 hours post-infection to a medium label virus-infected cells, the medium was changed 4 hours post-infection to a medium that was supplemented with [35S]Met (GE Healthcare). that was supplemented with [35S]Met (GE Healthcare).

Table 3. Recombinant IAVs used in study III. Table 3. Recombinant IAVs used in study III.

Recombinant virus name Backbone virus Description Recombinant virus name Backbone virus Description

A/WSN-NS1Mallard(wt) A/WSN/33 A/Mallard NS1 wt A/WSN-NS1Mallard(wt) A/WSN/33 A/Mallard NS1 wt

A/WSN-NS1Mallard(K217E) A/WSN/33 A/Mallard NS1 K217E (SH3-binding disrupting A/WSN-NS1Mallard(K217E) A/WSN/33 A/Mallard NS1 K217E (SH3-binding disrupting

mutation) mutation)

A/WSN-NS1WSN(wt) A/WSN/33 A/WSN NS1 wt A/WSN-NS1WSN(wt) A/WSN/33 A/WSN NS1 wt

A/WSN-NS1WSN(T215P) A/WSN/33 A/WSN NS1 T215P (SH3-binding competent A/WSN-NS1WSN(T215P) A/WSN/33 A/WSN NS1 T215P (SH3-binding competent

mutation) mutation)

3.4 Antibodies (I-III) 3.4 Antibodies (I-III) The following primary antibodies were used in this study: mouse anti-Myc (Sigma- The following primary antibodies were used in this study: mouse anti-Myc (Sigma- Aldrich), mouse anti-HA (Santa Cruz Biotechnology), mouse anti-α-tubulin (Sigma- Aldrich), mouse anti-HA (Santa Cruz Biotechnology), mouse anti-α-tubulin (Sigma- Aldrich), rabbit anti-Histone H3 (Cell Signaling Technologies), mouse anti-CrkL Aldrich), rabbit anti-Histone H3 (Cell Signaling Technologies), mouse anti-CrkL (Millipore), mouse anti-Crk (BD Transduction Laboratories), mouse anti-Akt (Cell (Millipore), mouse anti-Crk (BD Transduction Laboratories), mouse anti-Akt (Cell Signaling Technologies), rabbit anti-phospho Akt (Ser473) (Cell Signaling Technologies), Signaling Technologies), rabbit anti-phospho Akt (Ser473) (Cell Signaling Technologies), mouse anti-p85β (AbD Serotec) mouse anti-phosphotyrosine (PY20, Santa Cruz mouse anti-p85β (AbD Serotec) mouse anti-phosphotyrosine (PY20, Santa Cruz Biotechnologies), rabbit anti-NP (Ronni et al., 1995), and guinea-pig anti-NS1 (Melen et Biotechnologies), rabbit anti-NP (Ronni et al., 1995), and guinea-pig anti-NS1 (Melen et al., 2007). The secondary antibodies for Western blotting were purchased from LI-COR al., 2007). The secondary antibodies for Western blotting were purchased from LI-COR Biotechnology: IRDye680CW goat anti-mouse, IRDye800CW goat anti-mouse, IRDye680 Biotechnology: IRDye680CW goat anti-mouse, IRDye800CW goat anti-mouse, IRDye680 goat anti-rabbit, IRDye800CW goat anti-rabbit, IRDye800CW rabbit anti-guinea-pig, goat anti-rabbit, IRDye800CW goat anti-rabbit, IRDye800CW rabbit anti-guinea-pig, Streptavidin IRDye680CW, and Streptavidin IRDye800CW. AlexaFluor 488 goat anti- Streptavidin IRDye680CW, and Streptavidin IRDye800CW. AlexaFluor 488 goat anti- guinea-pig (Abcam) and AlexaFluor 546 anti-mouse (Molecular Probes) were used as guinea-pig (Abcam) and AlexaFluor 546 anti-mouse (Molecular Probes) were used as secondary antibodies for immuno fluorescence staining and nuclei were stained with secondary antibodies for immuno fluorescence staining and nuclei were stained with Hoechst (Sigma Aldrich). Hoechst (Sigma Aldrich).

3.5 Protein sequence alignment (I) 3.5 Protein sequence alignment (I) The amino acid sequences for different NS1 proteins were obtained from the NCBI The amino acid sequences for different NS1 proteins were obtained from the NCBI Influenza Virus Resource data base. The sequence alignment of multiple NS1 proteins Influenza Virus Resource data base. The sequence alignment of multiple NS1 proteins was done with ClustalW software. was done with ClustalW software.

40 40 3.6 Recombinant protein production, purification and phage screening 3.6 Recombinant protein production, purification and phage screening (I,II) (I,II) The fusion proteins with GST and MBP were expressed in E. coli strain BL21 and purified The fusion proteins with GST and MBP were expressed in E. coli strain BL21 and purified by using glutathione-Sepharose 4B agarose (GE Healthcare) or amylose resin (New by using glutathione-Sepharose 4B agarose (GE Healthcare) or amylose resin (New England Biolabs), respectively. The human proteome SH3 domain library used in this England Biolabs), respectively. The human proteome SH3 domain library used in this study is characterized and the phage screening was done as described (Kärkkäinen et study is characterized and the phage screening was done as described (Kärkkäinen et al., 2006). al., 2006).

3.7 Protein precipitation and Western blot (I-III) 3.7 Protein precipitation and Western blot (I-III) For immunoprecipitation and protein pulldown experiments, transfected or infected For immunoprecipitation and protein pulldown experiments, transfected or infected cells were collected and lysed in 1 % NP40 lysis buffer (150 mM NaCl; 50 mM Tris-HCl, cells were collected and lysed in 1 % NP40 lysis buffer (150 mM NaCl; 50 mM Tris-HCl, pH 7.9; 1 % NP40) in the presence of protease and phosphatase inhibitors. Cell lysates pH 7.9; 1 % NP40) in the presence of protease and phosphatase inhibitors. Cell lysates were used for immunoprecipitation with anti-CrkL (I-III) or anti-HA (II) antibodies were used for immunoprecipitation with anti-CrkL (I-III) or anti-HA (II) antibodies coupled to Dynabeads protein G magnetic beads (Invitrogen) or for streptavidin coupled to Dynabeads protein G magnetic beads (Invitrogen) or for streptavidin pulldown (I,II) with Tetralink tetrameric avidin resin (Promega) (I) or Dynabeads pulldown (I,II) with Tetralink tetrameric avidin resin (Promega) (I) or Dynabeads streptavidin coated magnetic beads (Invitrogen) (II). streptavidin coated magnetic beads (Invitrogen) (II). For total protein samples, the protein concentrations were determined by Bio-Rad For total protein samples, the protein concentrations were determined by Bio-Rad Protein Assay (Bio-Rad). Immunocomplexes and total protein samples were separated Protein Assay (Bio-Rad). Immunocomplexes and total protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membrane (Bio-Rad) for subsequent by SDS-PAGE and transferred onto nitrocellulose membrane (Bio-Rad) for subsequent immunoblotting with specific antibodies. Western blots were visualized with the immunoblotting with specific antibodies. Western blots were visualized with the Odyssey infrared imaging system (LI-COR Biosciences). Odyssey infrared imaging system (LI-COR Biosciences).

3.8 Detection of phosphorylated proteins (I-III) 3.8 Detection of phosphorylated proteins (I-III) To determine the phosphorylation status of Akt, Huh7 cells were transfected with To determine the phosphorylation status of Akt, Huh7 cells were transfected with different NS1 expression constructs (I-III) alone or together with Crk or CrkL expression different NS1 expression constructs (I-III) alone or together with Crk or CrkL expression constructs (II). Transfected cells were serum starved with a medium containing 2 % FBS constructs (II). Transfected cells were serum starved with a medium containing 2 % FBS for 12 hours prior the collection 48 hours post-transfection. Collected cells were lysed for 12 hours prior the collection 48 hours post-transfection. Collected cells were lysed with NP40 lysis buffer supplied with protease and phosphatase inhibitors and subjected with NP40 lysis buffer supplied with protease and phosphatase inhibitors and subjected to Western blotting with anti-pAkt (S473) antibody. To study the tyrosine to Western blotting with anti-pAkt (S473) antibody. To study the tyrosine phosphorylation of nuclear proteins (III), A549 cells were infected with recombinant phosphorylation of nuclear proteins (III), A549 cells were infected with recombinant IAVs for 24 hours. To enhance the accumulation of tyrosine phosphorylated proteins, IAVs for 24 hours. To enhance the accumulation of tyrosine phosphorylated proteins, cells were treated with phosphotyrosine phosphatase inhibitor pervanadate for 10 cells were treated with phosphotyrosine phosphatase inhibitor pervanadate for 10 minutes. The fractions were then prepared as described in section 3.10 and the lysates minutes. The fractions were then prepared as described in section 3.10 and the lysates were subjected to Western blot analysis with anti-pTyr antibody. were subjected to Western blot analysis with anti-pTyr antibody.

3.9 Dual-luciferase assays (I) 3.9 Dual-luciferase assays (I) ISRE-Luc reporter plasmid was transfected together with pcDNA-Renilla and NS1 ISRE-Luc reporter plasmid was transfected together with pcDNA-Renilla and NS1 expression constructs or empty vector into Huh7 cells. 22 hours after transfection, the expression constructs or empty vector into Huh7 cells. 22 hours after transfection, the cells were treated with 100 IU/ml of IFNβ (Betaferon, Schering) for 7 hours to induce the cells were treated with 100 IU/ml of IFNβ (Betaferon, Schering) for 7 hours to induce the reporter gene. The luciferase activities were measured using a dual-luciferase assay reporter gene. The luciferase activities were measured using a dual-luciferase assay (Promega). Renilla luciferase construct was used as an internal control for transfection. (Promega). Renilla luciferase construct was used as an internal control for transfection.

3.10 In vitro protein binding assays (I,II) 3.10 In vitro protein binding assays (I,II) Purified recombinant MBP-fusion proteins (I) were coated on 96-well plates (200 Purified recombinant MBP-fusion proteins (I) were coated on 96-well plates (200 ng/well) and blocked with 1.5 % BSA in TBS (Tris-buffered saline) for 1 hour. The wells ng/well) and blocked with 1.5 % BSA in TBS (Tris-buffered saline) for 1 hour. The wells were washed twice with TBST (TBS + 0.05 % Tween 20). Bacterial purified GST-PP-fused were washed twice with TBST (TBS + 0.05 % Tween 20). Bacterial purified GST-PP-fused

41 41 SH3 domains were added in 2-fold dilutions and incubated for 1.5 hours. After three SH3 domains were added in 2-fold dilutions and incubated for 1.5 hours. After three washes with TBST, the wells were incubated with streptavidin-biotinylated horseradish washes with TBST, the wells were incubated with streptavidin-biotinylated horseradish peroxidase complex (GE Healthcare) in TBS for 1 hour. The wells were again washed for peroxidase complex (GE Healthcare) in TBS for 1 hour. The wells were again washed for three times with TBST and 50 μl substrate reagent ABTS Single Solution (Invitrogen) was three times with TBST and 50 μl substrate reagent ABTS Single Solution (Invitrogen) was added. The absorbance was measured 20 minutes later at 405 nm. added. The absorbance was measured 20 minutes later at 405 nm. To examine the formation of the trimolecular complex of NS1, Crk and p85β in vitro, PP- To examine the formation of the trimolecular complex of NS1, Crk and p85β in vitro, PP- tagged p85β was purified from 293T transfected cells with Dynabeads streptavidin tagged p85β was purified from 293T transfected cells with Dynabeads streptavidin coated magnetic beads (Invitrogen) (II). Bacterial purified, GST-fused NS1 and CrkL-SH3 coated magnetic beads (Invitrogen) (II). Bacterial purified, GST-fused NS1 and CrkL-SH3 proteins were mixed together with purified p85β and the formed complexes were proteins were mixed together with purified p85β and the formed complexes were analyzed with Western blotting. analyzed with Western blotting.

3.11 Cell fractionation (III) 3.11 Cell fractionation (III) A549 cells were infected with influenza A viruses at an MOI 2 for 24 hours. The cells A549 cells were infected with influenza A viruses at an MOI 2 for 24 hours. The cells were scraped into 500 μl of ice cold Buffer A (20 mM Tris-HCl, pH7.5; 100 mM NaCl; 300 were scraped into 500 μl of ice cold Buffer A (20 mM Tris-HCl, pH7.5; 100 mM NaCl; 300 mM sucrose, 3 mM MgCl2) supplemented with 0.5 % Triton X-100 (Sigma Aldrich). After mM sucrose, 3 mM MgCl2) supplemented with 0.5 % Triton X-100 (Sigma Aldrich). After 10 minutes of incubation on ice, the nuclei were pelleted at 800xg for 10 minutes. The 10 minutes of incubation on ice, the nuclei were pelleted at 800xg for 10 minutes. The cytoplasmic fraction (C) was collected and was centrifuged for an additional 15 minutes cytoplasmic fraction (C) was collected and was centrifuged for an additional 15 minutes at 16100xg. The nuclear pellet was washed once with Buffer A +0.5 % Triton X-100 and at 16100xg. The nuclear pellet was washed once with Buffer A +0.5 % Triton X-100 and twice with Buffer A. The nuclear proteins were collected by dissolving the nuclear pellet twice with Buffer A. The nuclear proteins were collected by dissolving the nuclear pellet into Buffer B (20 mM Tris-Hcl, pH 8.0; 500 mM NaCl, 2 mM EDTA, pH8.0; 0.1 % NP40) into Buffer B (20 mM Tris-Hcl, pH 8.0; 500 mM NaCl, 2 mM EDTA, pH8.0; 0.1 % NP40) and sonicating for 3 seconds. The nuclear extract (N) was cleared by centrifugation at and sonicating for 3 seconds. The nuclear extract (N) was cleared by centrifugation at 16100xg for 15 minutes. 16100xg for 15 minutes.

3.12 Immunostaining and imaging (III) 3.12 Immunostaining and imaging (III) A549 cells grown on coverslips were infected with IAVs at an MOI of 0.5 for 20 hours. A549 cells grown on coverslips were infected with IAVs at an MOI of 0.5 for 20 hours. The cells were then fixed with ice cold methanol for 10 minutes at -20 °C and The cells were then fixed with ice cold methanol for 10 minutes at -20 °C and permeabilized with 0.1 % Triton X-100 for 10 minutes. The cells were blocked with 5 % permeabilized with 0.1 % Triton X-100 for 10 minutes. The cells were blocked with 5 % BSA and 10 % FBS in PBS. NS1 and CrkL were stained with guinea-pig anti-NS1 and mouse BSA and 10 % FBS in PBS. NS1 and CrkL were stained with guinea-pig anti-NS1 and mouse anti-CrkL antibodies, respectively, for 40 minutes in the blocking buffer. After three anti-CrkL antibodies, respectively, for 40 minutes in the blocking buffer. After three washes, the cells were incubated with secondary antibodies AlexaFluor 488 goat anti- washes, the cells were incubated with secondary antibodies AlexaFluor 488 goat anti- guinea pig or AlexaFluor 546 goat anti-mouse and washed again three times. The nuclei guinea pig or AlexaFluor 546 goat anti-mouse and washed again three times. The nuclei were stained with Hoechst for 5 minutes. The cells were visualized with Leica TCS SP8 were stained with Hoechst for 5 minutes. The cells were visualized with Leica TCS SP8 confocal microscope scanning the channels sequentially. Open source software Fiji was confocal microscope scanning the channels sequentially. Open source software Fiji was used to analyze the mean intensities of the CrkL fluorescence signal (Schindelin et al., used to analyze the mean intensities of the CrkL fluorescence signal (Schindelin et al., 2012). 2012).

42 42 4 RESULTS 4 RESULTS

4.1 Novel SH3 domain interaction partner for IAV NS1 protein (I-III) 4.1 Novel SH3 domain interaction partner for IAV NS1 protein (I-III)

4.1.1 Characterization of a novel interaction partner for NS1 protein (I-III) 4.1.1 Characterization of a novel interaction partner for NS1 protein (I-III) Sequence analysis of different viral proteins revealed that the NS1 protein of the Spanish Sequence analysis of different viral proteins revealed that the NS1 protein of the Spanish Flu IAV (A/Brevig Mission/1/18; A/Brevig) contains a potential class II SH3 domain Flu IAV (A/Brevig Mission/1/18; A/Brevig) contains a potential class II SH3 domain binding motif near the C-terminus of the protein sequence (see fig. 1A in I). The SH3 binding motif near the C-terminus of the protein sequence (see fig. 1A in I). The SH3 binding motif was noted to be very common among avian isolated IAVs but only binding motif was noted to be very common among avian isolated IAVs but only occasionally found in NS1 proteins of human IAVs (see fig. 1B in I). The A/Brevig and occasionally found in NS1 proteins of human IAVs (see fig. 1B in I). The A/Brevig and many avian isolated NS1 proteins contain a consensus sequence of PxɸPx+, whereas in many avian isolated NS1 proteins contain a consensus sequence of PxɸPx+, whereas in most human isolated IAV NS1 proteins the second proline has been substituted with most human isolated IAV NS1 proteins the second proline has been substituted with threonine (PxɸTPx+). In addition, this SH3 binding sequence motif is not present in the threonine (PxɸTPx+). In addition, this SH3 binding sequence motif is not present in the NS1 proteins of IAV strains commonly used in laboratory studies, such as A/WSN/33 NS1 proteins of IAV strains commonly used in laboratory studies, such as A/WSN/33 (H1N1), A/PR/8/34 (H1N1), and A/Udorn/72 (H3N2) (see fig 1B in I). (H1N1), A/PR/8/34 (H1N1), and A/Udorn/72 (H3N2) (see fig 1B in I). To identify possible SH3 interaction partners for NS1, we expressed the NS1 proteins of To identify possible SH3 interaction partners for NS1, we expressed the NS1 proteins of A/Brevig and A/Mallard as GST-fusion proteins and subjected them to affinity screening A/Brevig and A/Mallard as GST-fusion proteins and subjected them to affinity screening with human SH3 phage display library. An increased binding of SH3 domain clones to with human SH3 phage display library. An increased binding of SH3 domain clones to both NS1 proteins was observed as compared with the plain GST protein that was used both NS1 proteins was observed as compared with the plain GST protein that was used as a control. The identity of NS1-selected phagemids was determined by sequencing, as a control. The identity of NS1-selected phagemids was determined by sequencing, and more than 90 % of the phages contained the nSH3 domain of the adaptor proteins and more than 90 % of the phages contained the nSH3 domain of the adaptor proteins CrkII or CrkL. To confirm the interaction of NS1 with Crk SH3 domain found with phage CrkII or CrkL. To confirm the interaction of NS1 with Crk SH3 domain found with phage screening, we expressed recombinant GST-PP-fusion proteins of the nSH3 domains of screening, we expressed recombinant GST-PP-fusion proteins of the nSH3 domains of CrkII and CrkL. Since NS1 has been reported to interact with the SH3 domain of p85 CrkII and CrkL. Since NS1 has been reported to interact with the SH3 domain of p85 regulatory subunit of PI3K (Shin et al., 2007a), we also generated GST-PP-fusion proteins regulatory subunit of PI3K (Shin et al., 2007a), we also generated GST-PP-fusion proteins of SH3 domains of p85α and p85β. As a negative control, we used the SH3 domain of of SH3 domains of p85α and p85β. As a negative control, we used the SH3 domain of Eps8L1, which prefers ligands containing PXXDY-motif. The A/Mallard and A/Udorn NS1 Eps8L1, which prefers ligands containing PXXDY-motif. The A/Mallard and A/Udorn NS1 proteins were expressed as MBP-fusion proteins. In vitro protein binding assay with proteins were expressed as MBP-fusion proteins. In vitro protein binding assay with these proteins revealed the avid binding of the nSH3 domain of CrkII and CrkL to the these proteins revealed the avid binding of the nSH3 domain of CrkII and CrkL to the A/Mallard NS1 (see fig. 2A in I), which was in line with our phage screening result. In A/Mallard NS1 (see fig. 2A in I), which was in line with our phage screening result. In contrast, the A/Udorn NS1, which lacks the consensus SH3 binding motif, did not show contrast, the A/Udorn NS1, which lacks the consensus SH3 binding motif, did not show any measurable binding to the CrkII and CrkL SH3 domains (see fig. 2B in I). In addition, any measurable binding to the CrkII and CrkL SH3 domains (see fig. 2B in I). In addition, neither of the NS1 proteins showed binding to the SH3 domains of p85α, p85β, or to the neither of the NS1 proteins showed binding to the SH3 domains of p85α, p85β, or to the negative control Eps8L1 (see fig. 1A and B in I). negative control Eps8L1 (see fig. 1A and B in I). To study Crk-NS1 interaction with full-length proteins, we transfected PP-tagged (biotin To study Crk-NS1 interaction with full-length proteins, we transfected PP-tagged (biotin acceptor domain) expression constructs of CrkII and CrkL together with myc-tagged NS1 acceptor domain) expression constructs of CrkII and CrkL together with myc-tagged NS1 proteins of A/Brevig, A/Mallard, or A/Udorn. While A/Brevig and A/Mallard NS1 proteins of A/Brevig, A/Mallard, or A/Udorn. While A/Brevig and A/Mallard NS1 proteins readily co-precipitated with both CrkII and CrkL, A/Udorn NS1 failed to co- proteins readily co-precipitated with both CrkII and CrkL, A/Udorn NS1 failed to co- precipitate with either one of them (see fig. 3 in I). To further characterize the capacity precipitate with either one of them (see fig. 3 in I). To further characterize the capacity of all three Crk family members, CrkI, CrkII and CrkL, to interact with NS1 proteins, we of all three Crk family members, CrkI, CrkII and CrkL, to interact with NS1 proteins, we co-transfected different Crk-proteins together with A/Mallard NS1-wt or its SH3 binding co-transfected different Crk-proteins together with A/Mallard NS1-wt or its SH3 binding incompetent mutant (AxxA; P212A, P215A). We could not see any differences of the incompetent mutant (AxxA; P212A, P215A). We could not see any differences of the binding capacity of A/Mallard NS1 wt to CrkI, CrkII or CrkL, while the AxxA-mutation of binding capacity of A/Mallard NS1 wt to CrkI, CrkII or CrkL, while the AxxA-mutation of A/Mallard NS1 totally abrogated the binding (see fig. 1B in II). A/Mallard NS1 totally abrogated the binding (see fig. 1B in II). To extend these finding to IAV infection model, we used an avian IAV strain, A/Mallard To extend these finding to IAV infection model, we used an avian IAV strain, A/Mallard and a typical human IAV strain, A/Udorn to infect A549 cells. The NS1 protein from and a typical human IAV strain, A/Udorn to infect A549 cells. The NS1 protein from

43 43 A/Mallard infected cells readily co-precipitated with endogenous CrkL, whereas the NS1 A/Mallard infected cells readily co-precipitated with endogenous CrkL, whereas the NS1 protein from A/Udorn infected cells did not (see fig. 4 in I). In addition, we generated a protein from A/Udorn infected cells did not (see fig. 4 in I). In addition, we generated a set of recombinant viruses (see fig. 1A in III). The viruses express the NS1 protein from set of recombinant viruses (see fig. 1A in III). The viruses express the NS1 protein from either A/WSN or A/Mallard IAV strains. Mutation to the NS1 gene of A/Mallard (K217E) either A/WSN or A/Mallard IAV strains. Mutation to the NS1 gene of A/Mallard (K217E) generated an SH3-binding deficient NS1 protein, whereas the mutation T215P in A/WSN generated an SH3-binding deficient NS1 protein, whereas the mutation T215P in A/WSN NS1 gene transformed the A/WSN NS1 protein from SH3 binding incompetent to SH3 NS1 gene transformed the A/WSN NS1 protein from SH3 binding incompetent to SH3 binding competent. We immunoprecipated endogenous CrkL from cells infected with binding competent. We immunoprecipated endogenous CrkL from cells infected with these recombinant IAVs and examined NS1 co-precipitation by Western blotting. As these recombinant IAVs and examined NS1 co-precipitation by Western blotting. As expected based on our earlier results, A/Mallard NS1-wt was seen to co-precipitate with expected based on our earlier results, A/Mallard NS1-wt was seen to co-precipitate with CrkL, while the mutant, A/Mallard NS1-K217E, did not (see fig. 1B in III). Likewise, the CrkL, while the mutant, A/Mallard NS1-K217E, did not (see fig. 1B in III). Likewise, the mutant A/WSN NS1-T215P was observed to co-precipitate with CrkL, but the A/WSN-wt mutant A/WSN NS1-T215P was observed to co-precipitate with CrkL, but the A/WSN-wt was not (see fig. 1C in III). was not (see fig. 1C in III). To further characterize the critical amino acids involved in association of class II SH3 To further characterize the critical amino acids involved in association of class II SH3 binding motif containing NS1 with Crk proteins, we generated a series of point mutation binding motif containing NS1 with Crk proteins, we generated a series of point mutation variants of the binding site in A/Brevig NS1 (see fig. 5A in I). We transfected these mutant variants of the binding site in A/Brevig NS1 (see fig. 5A in I). We transfected these mutant variants into cells and performed immunoprecipitation of endogenous CrkL protein. variants into cells and performed immunoprecipitation of endogenous CrkL protein. While the wt A/Brevig NS1 co-precipitated with CrkL, the mutant variants with class II While the wt A/Brevig NS1 co-precipitated with CrkL, the mutant variants with class II binding motif disrupting mutation (M1; AxxA and M3; PxxPx-) could not be co- binding motif disrupting mutation (M1; AxxA and M3; PxxPx-) could not be co- precipitated with CrkL (see fig. 5B in I). Likewise, the mutation T215P (M5), mimicking precipitated with CrkL (see fig. 5B in I). Likewise, the mutation T215P (M5), mimicking the sequence of A/Udorn NS1, could not be co-precipitated with CrkL. In addition, the sequence of A/Udorn NS1, could not be co-precipitated with CrkL. In addition, swapping the lysine residue to arginine in the consensus sequence in A/Brevig NS1 (M4) swapping the lysine residue to arginine in the consensus sequence in A/Brevig NS1 (M4) diminished the binding to CrkL for ~50 %, which is in agreement with the binding diminished the binding to CrkL for ~50 %, which is in agreement with the binding preference of nSH3 domain of Crk proteins to sequences with lysine as a positively preference of nSH3 domain of Crk proteins to sequences with lysine as a positively charged residue (Knudsen et al., 1995). charged residue (Knudsen et al., 1995).

4.1.2 Functional role for the Crk-NS1 interaction (I,II) 4.1.2 Functional role for the Crk-NS1 interaction (I,II) To study the biological relevance of Crk SH3-NS1 interaction, we tested the activities of To study the biological relevance of Crk SH3-NS1 interaction, we tested the activities of A/Brevig and A/Mallard NS1 proteins and their SH3 binding-deficient mutants in two A/Brevig and A/Mallard NS1 proteins and their SH3 binding-deficient mutants in two different cellular functions found in the literature for NS1 protein. To test the ability of different cellular functions found in the literature for NS1 protein. To test the ability of these NS1 proteins to repress the expression of an interferon-stimulated gene, we co- these NS1 proteins to repress the expression of an interferon-stimulated gene, we co- transfected cells with the ISRE-Luc (interferon-stimulated response element-containing transfected cells with the ISRE-Luc (interferon-stimulated response element-containing fragment) reporter plasmid together with an empty control expression vector or vector fragment) reporter plasmid together with an empty control expression vector or vector expressing NS1 of A/Brevig, A/Mallard, or A/Udorn, or the SH3-binding mutant versions expressing NS1 of A/Brevig, A/Mallard, or A/Udorn, or the SH3-binding mutant versions of A/Brevig (A/Brevig NS1-AxxA) and A/Mallard (A/Mallard NS1-AxxA). After the of A/Brevig (A/Brevig NS1-AxxA) and A/Mallard (A/Mallard NS1-AxxA). After the induction of the reporter vector with IFNβ, the luciferase activity was measured. All NS1 induction of the reporter vector with IFNβ, the luciferase activity was measured. All NS1 proteins, including the SH3-binding mutants of A/Brevig and A/Mallard were able to proteins, including the SH3-binding mutants of A/Brevig and A/Mallard were able to inhibit the luciferase expression from ISRE-Luc reporter vector compared to the control inhibit the luciferase expression from ISRE-Luc reporter vector compared to the control where an empty expression vector was transfected (see fig. 6A in I). This indicated that where an empty expression vector was transfected (see fig. 6A in I). This indicated that the binding of NS1 to Crk and CrkL SH3 domains was not required for the capacity of IAV the binding of NS1 to Crk and CrkL SH3 domains was not required for the capacity of IAV NS1 proteins to inhibit interferon-stimulated gene expression. NS1 proteins to inhibit interferon-stimulated gene expression. To test the effect of the Crk-NS1 interaction to another known function for NS1, the To test the effect of the Crk-NS1 interaction to another known function for NS1, the activation of PI3K pathway, we transfected the same set of NS1 proteins and their activation of PI3K pathway, we transfected the same set of NS1 proteins and their mutants and analyzed the ability of these proteins to activate the PI3K signaling by mutants and analyzed the ability of these proteins to activate the PI3K signaling by determining the phosphorylation status of Akt (pAkt S473) by Western blotting. The determining the phosphorylation status of Akt (pAkt S473) by Western blotting. The levels of pAkt were significantly higher in cells transfected with A/Brevig or A/Mallard levels of pAkt were significantly higher in cells transfected with A/Brevig or A/Mallard NS1-wt compared with the NS1 constructs lacking the SH3 binding motif either naturally, NS1-wt compared with the NS1 constructs lacking the SH3 binding motif either naturally,

44 44 as in A/Udorn and A/WSN or by mutation A/Brevig NS1-AxxA and A/Mallard NS1-AxxA as in A/Udorn and A/WSN or by mutation A/Brevig NS1-AxxA and A/Mallard NS1-AxxA (see fig. 6B in I). Thus, the SH3 binding motif provides the NS1 with enhanced capacity (see fig. 6B in I). Thus, the SH3 binding motif provides the NS1 with enhanced capacity to induce PI3K activation compared to NS1 proteins lacking the binding site. to induce PI3K activation compared to NS1 proteins lacking the binding site. Tyrosine residue at position 89 in NS1 has been reported to be critical for NS1- p85β Tyrosine residue at position 89 in NS1 has been reported to be critical for NS1- p85β interaction, and NS1-Y89F mutant is not able to interact with p85β or to activate PI3K interaction, and NS1-Y89F mutant is not able to interact with p85β or to activate PI3K signaling (Hale et al., 2006). To further analyze the amino acid residues important for signaling (Hale et al., 2006). To further analyze the amino acid residues important for the enhanced PI3K activation by SH3 binding competent NS1 proteins, we generated the enhanced PI3K activation by SH3 binding competent NS1 proteins, we generated Y89F-mutant construct of A/Mallard NS1. Cells were transfected with A/Mallard NS1- Y89F-mutant construct of A/Mallard NS1. Cells were transfected with A/Mallard NS1- wt, NS1-AxxA (SH3 binding incompetent mutant), or NS1-Y89F (p85β binding mutant), wt, NS1-AxxA (SH3 binding incompetent mutant), or NS1-Y89F (p85β binding mutant), and the phosphorylation status of Akt was examined. While the expression of A/Mallard and the phosphorylation status of Akt was examined. While the expression of A/Mallard NS1-AxxA showed reduced PI3K activation compared to the NS1-wt, the Y89F mutation NS1-AxxA showed reduced PI3K activation compared to the NS1-wt, the Y89F mutation totally abrogated the induction (see fig. 2A in II). Thus, while the SH3 binding capacity totally abrogated the induction (see fig. 2A in II). Thus, while the SH3 binding capacity potentiates the PI3K activation by NS1, the binding of NS1 to p85β through Y89 is potentiates the PI3K activation by NS1, the binding of NS1 to p85β through Y89 is indispensable for the activation. indispensable for the activation.

4.1.3 Trimeric complex formation upon Crk-NS1 interaction (II) 4.1.3 Trimeric complex formation upon Crk-NS1 interaction (II) In addition to the Crk-NS1 interaction reported here, both NS1 and Crk have been In addition to the Crk-NS1 interaction reported here, both NS1 and Crk have been described to interact with p85 (see fig. 3A in II). The Crk-p85 interaction has been described to interact with p85 (see fig. 3A in II). The Crk-p85 interaction has been reported to be mediated by the Crk nSH3 domain (Gelkop et al., 2001; Sattler et al., reported to be mediated by the Crk nSH3 domain (Gelkop et al., 2001; Sattler et al., 1997), the same SH3 domain that we have described to mediate the Crk-NS1 interaction. 1997), the same SH3 domain that we have described to mediate the Crk-NS1 interaction. To study whether these three proteins are in the same complex, we performed To study whether these three proteins are in the same complex, we performed immunoprecipitation of endogenous CrkL from cells transfected with A/Mallard NS1-wt, immunoprecipitation of endogenous CrkL from cells transfected with A/Mallard NS1-wt, NS1-AxxA, or NS1-Y89F. In agreement with our earlier results, the A/Mallard NS1 protein NS1-AxxA, or NS1-Y89F. In agreement with our earlier results, the A/Mallard NS1 protein readily co-precipitated with CrkL when the SH3 binding motif was intact (NS1-wt and readily co-precipitated with CrkL when the SH3 binding motif was intact (NS1-wt and NS1-Y89F), but binding was abrogated when the SH3 binding site was mutated (NS1- NS1-Y89F), but binding was abrogated when the SH3 binding site was mutated (NS1- AxxA) (see fig. 2B in II). p85β was seen to co-precipitate with CrkL when NS1 was not AxxA) (see fig. 2B in II). p85β was seen to co-precipitate with CrkL when NS1 was not expressed. The expression of NS1-wt or NS1-AxxA did not affect the net association of expressed. The expression of NS1-wt or NS1-AxxA did not affect the net association of p85β with CrkL. However, transfection of NS1-Y89F potently inhibited the association of p85β with CrkL. However, transfection of NS1-Y89F potently inhibited the association of p85β with CrkL. Since the binding of p85β to Crk is mediated by the nSH3 domain of Crk, p85β with CrkL. Since the binding of p85β to Crk is mediated by the nSH3 domain of Crk, we hypothesized that NS1 could displace p85β from binding to the nSH3 domain of Crk we hypothesized that NS1 could displace p85β from binding to the nSH3 domain of Crk proteins. proteins. To demonstrate that NS1 could disrupt the preformed p85β-CrkL complex, we co- To demonstrate that NS1 could disrupt the preformed p85β-CrkL complex, we co- transfected cells with p85β together with CrkL. The lysates were mixed with increasing transfected cells with p85β together with CrkL. The lysates were mixed with increasing amounts of recombinant GST-NS1-Y89F of A/Mallard, or NS1-AxxA as a control. We amounts of recombinant GST-NS1-Y89F of A/Mallard, or NS1-AxxA as a control. We examined the amounts of p85β that co-precipitated with CrkL by Western blotting. examined the amounts of p85β that co-precipitated with CrkL by Western blotting. Recombinant NS1-Y89F was able to displace p85β from its complex with CrkL in a dose- Recombinant NS1-Y89F was able to displace p85β from its complex with CrkL in a dose- dependent manner (see fig. 2C in II). To further prove that NS1 is able to displace p85β dependent manner (see fig. 2C in II). To further prove that NS1 is able to displace p85β from a preformed complex with CrkL, we used a mutant construct of p85β (V573M), from a preformed complex with CrkL, we used a mutant construct of p85β (V573M), which prevents the NS1-p85β interaction, and recombinant A/Mallard NS1-wt protein which prevents the NS1-p85β interaction, and recombinant A/Mallard NS1-wt protein in a similar experiment as above. We obtained a similar result, where NS1-wt could in a similar experiment as above. We obtained a similar result, where NS1-wt could displace p85β-V573M from CrkL in a dose-dependent manner (see supplementary fig. 1 displace p85β-V573M from CrkL in a dose-dependent manner (see supplementary fig. 1 in II). From these results and the already known interactions of these proteins (see fig. in II). From these results and the already known interactions of these proteins (see fig. 3A in II), we hypothesized that two alternative trimeric complexes could be formed (see 3A in II), we hypothesized that two alternative trimeric complexes could be formed (see fig. 3B in II). The interaction between CrkL and p85β may be indirectly mediated via the fig. 3B in II). The interaction between CrkL and p85β may be indirectly mediated via the dual capacity of NS1 to bind both p85β and CrkL when SH3 binding-competent NS1 is dual capacity of NS1 to bind both p85β and CrkL when SH3 binding-competent NS1 is expressed (Complex 1). On the other hand, we also hypothesized, that SH3 binding expressed (Complex 1). On the other hand, we also hypothesized, that SH3 binding

45 45 deficient NS1 could form a complex with CrkL through association with p85β (Complex deficient NS1 could form a complex with CrkL through association with p85β (Complex 2). The modest affinity of CrkL nSH3 to p85β and/or low abundance of p85β compared 2). The modest affinity of CrkL nSH3 to p85β and/or low abundance of p85β compared to CrkL and NS1 could explain why we cannot detect the association of SH3 binding to CrkL and NS1 could explain why we cannot detect the association of SH3 binding deficient NS1 with CrkL. deficient NS1 with CrkL. To test our hypothesis, we co-transfected increasing amounts of p85β together with a To test our hypothesis, we co-transfected increasing amounts of p85β together with a constant amount of A/Mallard NS1-wt or NS1-AxxA. Co-precipitation of NS1-wt with constant amount of A/Mallard NS1-wt or NS1-AxxA. Co-precipitation of NS1-wt with CrkL was already detectable in the absence of p85β transfection, and the overexpression CrkL was already detectable in the absence of p85β transfection, and the overexpression of p85β did not affect the association (see fig. 4A in II). In contrast, increasing amounts of p85β did not affect the association (see fig. 4A in II). In contrast, increasing amounts of p85β enabled the CrkL-NS1-AxxA co-precipitation in a dose-dependent manner. of p85β enabled the CrkL-NS1-AxxA co-precipitation in a dose-dependent manner. Likewise, co-precipitation of naturally SH3 binding-incompetent NS1 proteins, A/Udorn Likewise, co-precipitation of naturally SH3 binding-incompetent NS1 proteins, A/Udorn and A/WSN, with CrkL was evident when p85β was overexpressed (see fig. 4B in II). In and A/WSN, with CrkL was evident when p85β was overexpressed (see fig. 4B in II). In strong support for our hypothesis of two alternative complexes, the ability of CrkL to strong support for our hypothesis of two alternative complexes, the ability of CrkL to precipitate NS1 proteins of A/Udorn and A/WSN through p85β was abrogated by the precipitate NS1 proteins of A/Udorn and A/WSN through p85β was abrogated by the p85β-(P294A, P297A) mutation, which impairs the Crk-p85β interaction. Thus, CrkL- p85β-(P294A, P297A) mutation, which impairs the Crk-p85β interaction. Thus, CrkL- p85β-NS1 complex (Complex 2) may exist when an SH3 binding-incompetent NS1 is p85β-NS1 complex (Complex 2) may exist when an SH3 binding-incompetent NS1 is expressed and it is mediated through the p85β interaction with Crk-SH3 domain. expressed and it is mediated through the p85β interaction with Crk-SH3 domain. However, assembly of Complex 2, where p85β is bridging the interaction between CrkL However, assembly of Complex 2, where p85β is bridging the interaction between CrkL and SH3 binding deficient NS1, could only be seen when p85β was overexpressed. Thus, and SH3 binding deficient NS1, could only be seen when p85β was overexpressed. Thus, the recruitment of CrkL to p85β-NS1 complexes via p85β seems to be less efficient than the recruitment of CrkL to p85β-NS1 complexes via p85β seems to be less efficient than recruitment via an SH3 domain mediated CrkL-NS1 interaction. recruitment via an SH3 domain mediated CrkL-NS1 interaction. To further study the model of two alternative trimeric complexes, we made an To further study the model of two alternative trimeric complexes, we made an experiment where A/Mallard NS1-wt, -AxxA or -Y89F were co-transfected with p85β wt, experiment where A/Mallard NS1-wt, -AxxA or -Y89F were co-transfected with p85β wt, or its mutantsP294A/ P297A, and V573M in all relevant combinations. or its mutantsP294A/ P297A, and V573M in all relevant combinations. To further study the model of two alternative trimeric complexes, we made an To further study the model of two alternative trimeric complexes, we made an experiment where A/Mallard NS1-wt, -AxxA or -Y89F were co-transfected with p85β wt, experiment where A/Mallard NS1-wt, -AxxA or -Y89F were co-transfected with p85β wt, or its mutants P294A, P297A and V573M in all relevant combinations. The p85β-wt could or its mutants P294A, P297A and V573M in all relevant combinations. The p85β-wt could be co-precipitated with CrkL from cells co-transfected with NS1-wt and NS1-AxxA, be co-precipitated with CrkL from cells co-transfected with NS1-wt and NS1-AxxA, whereas expression of NS1-Y89F inhibited this association, which was in line with the whereas expression of NS1-Y89F inhibited this association, which was in line with the earlier results (see fig. 5, panel II, lanes 3-5 in II). p85β-V573M, the NS1-binding deficient earlier results (see fig. 5, panel II, lanes 3-5 in II). p85β-V573M, the NS1-binding deficient mutant, had no effect on binding to CrkL but reduced the p85β-CrkL association in cells mutant, had no effect on binding to CrkL but reduced the p85β-CrkL association in cells transfected with NS1-wt and NS1-Y89F (lanes 10 and 13). The expression of p85β- transfected with NS1-wt and NS1-Y89F (lanes 10 and 13). The expression of p85β- (P294A, P297A), the SH3 binding site mutant, abolished the p85β-CrkL binding (lane 6). (P294A, P297A), the SH3 binding site mutant, abolished the p85β-CrkL binding (lane 6). This association could be rescued by expression of NS1-wt (lane 7) but not of NS1-AxxA This association could be rescued by expression of NS1-wt (lane 7) but not of NS1-AxxA (lane 8) or NS1-Y89F (lane 9). In addition, NS1-wt increased the association of p85β and (lane 8) or NS1-Y89F (lane 9). In addition, NS1-wt increased the association of p85β and CrkL (see fig. 5, panel VI, lanes 3 and 7 in II). These results confirm that NS1 can serve as CrkL (see fig. 5, panel VI, lanes 3 and 7 in II). These results confirm that NS1 can serve as a bridging factor between p85β and CrkL in Complex 1 (see fig. 3B in II). a bridging factor between p85β and CrkL in Complex 1 (see fig. 3B in II). To confirm that the trimeric complex of NS1, p85β and CrkL can assemble in the absence To confirm that the trimeric complex of NS1, p85β and CrkL can assemble in the absence of additional cellular factors, we expressed and purified full-length p85β-wt, p85β- of additional cellular factors, we expressed and purified full-length p85β-wt, p85β- (P294A, P297A), NS1-wt and NS1-AxxA proteins, as well as the nSH3 domain fragment (P294A, P297A), NS1-wt and NS1-AxxA proteins, as well as the nSH3 domain fragment of CrkL. The recombinant proteins were mixed in different combinations and proteins of CrkL. The recombinant proteins were mixed in different combinations and proteins associated with p85β were precipitated. p85β-(P294A, P297A) could associate with CrkL- associated with p85β were precipitated. p85β-(P294A, P297A) could associate with CrkL- nSH3 only when NS1-wt was present, attesting that Complex 1 could be assembled in nSH3 only when NS1-wt was present, attesting that Complex 1 could be assembled in vitro from purified recombinant proteins (see supplementary fig. 2A in II). The SH3 vitro from purified recombinant proteins (see supplementary fig. 2A in II). The SH3 binding-deficient NS1-AxxA could associate with CrkL-nSH3 only when p85β-wt was binding-deficient NS1-AxxA could associate with CrkL-nSH3 only when p85β-wt was present, resulting in the assembly of Complex 2. p85β with the SH3 binding site mutation present, resulting in the assembly of Complex 2. p85β with the SH3 binding site mutation (P294A, P297A) could not mediate the formation of the trimeric complex. (P294A, P297A) could not mediate the formation of the trimeric complex.

46 46 4.1.4 Functional consequences of the trimeric complex (II) 4.1.4 Functional consequences of the trimeric complex (II) Based on results concerning the rearrangement of the p85β-CrkL complex by NS1, we Based on results concerning the rearrangement of the p85β-CrkL complex by NS1, we reasoned that the lower PI3K induction by SH3 binding deficient NS1 proteins could be reasoned that the lower PI3K induction by SH3 binding deficient NS1 proteins could be rescued by overexpression of Crk proteins. To test the idea, we transfected A/Mallard rescued by overexpression of Crk proteins. To test the idea, we transfected A/Mallard NS1-wt, NS1-AxxA and A/Udorn NS1 with or without CrkL. Overexpression of CrkL had a NS1-wt, NS1-AxxA and A/Udorn NS1 with or without CrkL. Overexpression of CrkL had a modest enhancing effect on NS1-wt induced Akt phosphorylation (see fig. 6A and B in modest enhancing effect on NS1-wt induced Akt phosphorylation (see fig. 6A and B in II), while the weaker PI3K activation by NS1-AxxA was significantly enhanced by CrkL II), while the weaker PI3K activation by NS1-AxxA was significantly enhanced by CrkL overexpression. A similar potentiation of PI3K activation by A/Udorn NS1 was observed overexpression. A similar potentiation of PI3K activation by A/Udorn NS1 was observed when CrkL was overexpressed. when CrkL was overexpressed. The SH3 binding function of NS1 has been reported to regulate the activity of c-Abl The SH3 binding function of NS1 has been reported to regulate the activity of c-Abl (Hrincius et al., 2014) Moreover c-Abl phosphorylates p85β (Sattler et al., 1996). We (Hrincius et al., 2014) Moreover c-Abl phosphorylates p85β (Sattler et al., 1996). We tested whether c-Abl has a role in regulation of PI3K activation by SH3 binding tested whether c-Abl has a role in regulation of PI3K activation by SH3 binding competent NS1 proteins. The inhibition of c-Abl with a potent c-Abl inhibitor, imatinib, competent NS1 proteins. The inhibition of c-Abl with a potent c-Abl inhibitor, imatinib, suppressed the tyrosine phosphorylation of CrkL but did not have an effect on NS1- suppressed the tyrosine phosphorylation of CrkL but did not have an effect on NS1- induced Akt phosphorylation (see supplementary fig. 3 in II). Thus, c-Abl does not seem induced Akt phosphorylation (see supplementary fig. 3 in II). Thus, c-Abl does not seem to have a critical role in this regulation. to have a critical role in this regulation.

4.2 Localization of the Crk-NS1 complex (III) 4.2 Localization of the Crk-NS1 complex (III)

4.2.1 NS1 mediates nuclear translocation of Crk proteins (III) 4.2.1 NS1 mediates nuclear translocation of Crk proteins (III) To study the localization of NS1 and Crk proteins in IAV infected cells, we analyzed cells To study the localization of NS1 and Crk proteins in IAV infected cells, we analyzed cells infected with recombinant IAVs (see Table 3) by immunostaining and confocal imaging. infected with recombinant IAVs (see Table 3) by immunostaining and confocal imaging. A/Mallard NS1-wt and NS1-K217E proteins were predominantly seen to localize in the A/Mallard NS1-wt and NS1-K217E proteins were predominantly seen to localize in the nucleus of infected cells (see fig. 2A in III). In mock-infected cells, CrkL was mainly nucleus of infected cells (see fig. 2A in III). In mock-infected cells, CrkL was mainly localized in the cytoplasm, while in A/Mallard NS1-wt expressing cells CrkL signal was localized in the cytoplasm, while in A/Mallard NS1-wt expressing cells CrkL signal was observed to co-localize with NS1 signal in the nucleus. In contrast, the localization of observed to co-localize with NS1 signal in the nucleus. In contrast, the localization of CrkL in A/Mallard NS1-K217E (SH3 binding deficient mutation) expressing cells did not CrkL in A/Mallard NS1-K217E (SH3 binding deficient mutation) expressing cells did not differ from mock-infected cells. Statistical analysis of CrkL nuclear signal confirmed that differ from mock-infected cells. Statistical analysis of CrkL nuclear signal confirmed that the distribution of Crk proteins was strikingly different in A/WSN-NS1Mallard(wt) –infected the distribution of Crk proteins was strikingly different in A/WSN-NS1Mallard(wt) –infected cells compared to A/WSN-NS1Mallard(K217E) –infected or mock-infected cells (see fig. 2B in cells compared to A/WSN-NS1Mallard(K217E) –infected or mock-infected cells (see fig. 2B in III). To confirm the translocation of Crk proteins seen with imaging studies, we prepared III). To confirm the translocation of Crk proteins seen with imaging studies, we prepared cytoplasmic and nuclear fractions of cells infected with recombinant viruses. Strong cytoplasmic and nuclear fractions of cells infected with recombinant viruses. Strong signals of CrkI, CrkII and CrkL proteins could be detected from the cytoplasmic fractions signals of CrkI, CrkII and CrkL proteins could be detected from the cytoplasmic fractions (C) of mock infected cells (see fig. 3A and B in III), whereas nuclear fractions (N) did not (C) of mock infected cells (see fig. 3A and B in III), whereas nuclear fractions (N) did not contain any detectable amounts of these proteins. The distribution of Crk proteins in contain any detectable amounts of these proteins. The distribution of Crk proteins in cells infected with viruses expressing an SH3 binding deficient NS1 protein cells infected with viruses expressing an SH3 binding deficient NS1 protein (NS1Mallard(K217E) or NS1WSN(wt)) was identical to mock-infected cells. Remarkably, in cells (NS1Mallard(K217E) or NS1WSN(wt)) was identical to mock-infected cells. Remarkably, in cells infected with viruses that contain an SH3 binding competent NS1 (NS1Mallard(wt) or infected with viruses that contain an SH3 binding competent NS1 (NS1Mallard(wt) or NS1WSN(T215P)) all Crk proteins were found in the nuclear fractions, especially CrkL. Thus, NS1WSN(T215P)) all Crk proteins were found in the nuclear fractions, especially CrkL. Thus, the SH3 binding motif of NS1 mediates the translocation of Crk proteins into the nucleus the SH3 binding motif of NS1 mediates the translocation of Crk proteins into the nucleus in IAV infected cells. in IAV infected cells.

4.2.2 Functional role of Crk translocation into the nucleus by NS1 (III) 4.2.2 Functional role of Crk translocation into the nucleus by NS1 (III) To study whether the NS1-SH3 domain binding mediated translocation of Crk proteins To study whether the NS1-SH3 domain binding mediated translocation of Crk proteins into the nucleus is responsible for enhancing the NS1-induced PI3K activation, we into the nucleus is responsible for enhancing the NS1-induced PI3K activation, we constructed an A/Mallard NS1 protein, which remains predominantly in the cytoplasm constructed an A/Mallard NS1 protein, which remains predominantly in the cytoplasm (NS1-Cyto). CrkL was observed to co-localize with NS1-Cyto in the cytoplasm in (NS1-Cyto). CrkL was observed to co-localize with NS1-Cyto in the cytoplasm in

47 47 transfected cells (see fig. 4A in III). However, the NS1-Cyto was able to induce the transfected cells (see fig. 4A in III). However, the NS1-Cyto was able to induce the phosphorylation of Akt in similar level as NS1-wt (see fig. 4B in III). Thus, the NS1- phosphorylation of Akt in similar level as NS1-wt (see fig. 4B in III). Thus, the NS1- mediated translocation of Crk proteins is not involved in the activation of PI3K. mediated translocation of Crk proteins is not involved in the activation of PI3K. Since Crk proteins are involved in the regulation of many tyrosine kinases, we next Since Crk proteins are involved in the regulation of many tyrosine kinases, we next investigated, whether the nuclear translocation of these proteins by NS1 would have an investigated, whether the nuclear translocation of these proteins by NS1 would have an effect on tyrosine phosphorylation of cellular proteins. Cells infected with recombinant effect on tyrosine phosphorylation of cellular proteins. Cells infected with recombinant viruses were subjected to cytoplasmic and nuclear fractionation. A new, approximately viruses were subjected to cytoplasmic and nuclear fractionation. A new, approximately 135 kDa tyrosine phosphorylated protein could be observed from the nuclear fractions 135 kDa tyrosine phosphorylated protein could be observed from the nuclear fractions of cells infected with viruses having SH3 binding competent NS1 (NS1Mallard(wt) or of cells infected with viruses having SH3 binding competent NS1 (NS1Mallard(wt) or NS1WSN(T215P)) (see fig. 5 in III). This phosphotyrosine-containing protein was not NS1WSN(T215P)) (see fig. 5 in III). This phosphotyrosine-containing protein was not detectable from the nuclear fractions of cells infected with viruses containing SH3 detectable from the nuclear fractions of cells infected with viruses containing SH3 binding deficient NS1 protein (NS1Mallard(K217E) or NS1WSN(wt)) or mock-infected cells. Thus, binding deficient NS1 protein (NS1Mallard(K217E) or NS1WSN(wt)) or mock-infected cells. Thus, we concluded that the nuclear translocation of Crk proteins by NS1 results in novel we concluded that the nuclear translocation of Crk proteins by NS1 results in novel tyrosine phosphorylation of a nuclear protein. tyrosine phosphorylation of a nuclear protein.

48 48 5 DISCUSSION 5 DISCUSSION

The NS1 protein of IAV is the key factor regulating the host cell responses to facilitate The NS1 protein of IAV is the key factor regulating the host cell responses to facilitate efficient virus replication. It is also considered to be a major virulence factor in IAV efficient virus replication. It is also considered to be a major virulence factor in IAV infected cells. It inhibits the host cell IFN and antiviral responses by blocking the infected cells. It inhibits the host cell IFN and antiviral responses by blocking the activation of RIG-I and preventing the processing of cellular mRNA. NS1 also inhibits activation of RIG-I and preventing the processing of cellular mRNA. NS1 also inhibits specific antiviral effector proteins such as OAS and PKR, and modulates apoptosis. In specific antiviral effector proteins such as OAS and PKR, and modulates apoptosis. In addition, the binding of NS1 to p85β regulatory subunit of PI3K and the subsequent addition, the binding of NS1 to p85β regulatory subunit of PI3K and the subsequent activation of PI3K/Akt pathway is conserved among NS1 proteins of diverse IAV strains activation of PI3K/Akt pathway is conserved among NS1 proteins of diverse IAV strains (Ehrhardt and Ludwig, 2009; Hale and Randall, 2007). The IAV NS1 protein has been (Ehrhardt and Ludwig, 2009; Hale and Randall, 2007). The IAV NS1 protein has been reported to have numerous functions and host cell interaction partners in the infected reported to have numerous functions and host cell interaction partners in the infected cell (Ayllon and Garcia-Sastre, 2015). Despite the wealth of such previous data on NS1 cell (Ayllon and Garcia-Sastre, 2015). Despite the wealth of such previous data on NS1 and its cellular partners, the exact mechanism by which NS1 acts as an IAV virulence and its cellular partners, the exact mechanism by which NS1 acts as an IAV virulence factor, and especially NS1 functions beyond its role as an antagonist of interferon factor, and especially NS1 functions beyond its role as an antagonist of interferon signaling, have remained elusive. In studies reported in this thesis we investigated novel signaling, have remained elusive. In studies reported in this thesis we investigated novel host cell proteins that could be involved in NS1 mediated pathogenesis of different IAV host cell proteins that could be involved in NS1 mediated pathogenesis of different IAV strains. strains.

5.1 Crk-NS1 interaction and PI3K activation 5.1 Crk-NS1 interaction and PI3K activation Many viral proteins have evolved an SH3 binding motif in order to exploit host SH3 Many viral proteins have evolved an SH3 binding motif in order to exploit host SH3 domain containing proteins to support virus growth and replication. In this thesis, we domain containing proteins to support virus growth and replication. In this thesis, we focused on IAV-host cell interactions mediated by SH3 domains. The sequence analysis focused on IAV-host cell interactions mediated by SH3 domains. The sequence analysis of NS1 proteins of different IAV strains revealed that the NS1 protein of Spanish Flu and of NS1 proteins of different IAV strains revealed that the NS1 protein of Spanish Flu and many avian IAV isolates contain an SH3 binding motif, which was not found in most of many avian IAV isolates contain an SH3 binding motif, which was not found in most of the seasonal human IAV strains. To identify new host SH3 interaction partners for the the seasonal human IAV strains. To identify new host SH3 interaction partners for the NS1 protein, we used a phage display library that contains virtually all human SH3 NS1 protein, we used a phage display library that contains virtually all human SH3 domain sequences. We were able to show that this motif mediates avid and specific domain sequences. We were able to show that this motif mediates avid and specific binding to the nSH3 domain of closely related Crk family adaptor proteins. We also binding to the nSH3 domain of closely related Crk family adaptor proteins. We also demonstrated that the Crk-SH3 binding motif provided the NS1 proteins with enhanced demonstrated that the Crk-SH3 binding motif provided the NS1 proteins with enhanced capacity to activate the PI3K/Akt signaling compared to the NS1 proteins which lack the capacity to activate the PI3K/Akt signaling compared to the NS1 proteins which lack the SH3 binding site. The Crk proteins have been reported to interact with the p85 SH3 binding site. The Crk proteins have been reported to interact with the p85 regulatory subunit of PI3K and to be involved in the activation of the pathway (Akagi et regulatory subunit of PI3K and to be involved in the activation of the pathway (Akagi et al., 2000; Gelkop et al., 2001; Sattler et al., 1996). Since both p85β and NS1 bind to the al., 2000; Gelkop et al., 2001; Sattler et al., 1996). Since both p85β and NS1 bind to the same nSH3 domain of Crk proteins, we further sought to examine the biochemical same nSH3 domain of Crk proteins, we further sought to examine the biochemical mechanism responsible for the superactivation of PI3K/Akt signaling by SH3 binding mechanism responsible for the superactivation of PI3K/Akt signaling by SH3 binding competent NS1 proteins. We demonstrated that NS1 proteins with SH3 binding capacity competent NS1 proteins. We demonstrated that NS1 proteins with SH3 binding capacity can disrupt naturally occurring p85-Crk interaction, by competing with p85 for binding can disrupt naturally occurring p85-Crk interaction, by competing with p85 for binding to the Crk nSH3 domain. This was shown to result in rearrangement of the Crk-p85β to the Crk nSH3 domain. This was shown to result in rearrangement of the Crk-p85β complex and in the assembly of a novel trimeric complex of Crk-NS1-p85β (Complex 1) complex and in the assembly of a novel trimeric complex of Crk-NS1-p85β (Complex 1) where NS1 is the bridging factor. We also noticed that when SH3 binding incompetent where NS1 is the bridging factor. We also noticed that when SH3 binding incompetent NS1 is expressed, an alternative trimeric complex is formed, where instead of NS1, the NS1 is expressed, an alternative trimeric complex is formed, where instead of NS1, the p85β serves as the bridging factor by binding to both NS1 and nSH3 domain of Crk. This p85β serves as the bridging factor by binding to both NS1 and nSH3 domain of Crk. This leads to the formation of NS1-p85β-Crk complex (Complex 2). Because subcellular leads to the formation of NS1-p85β-Crk complex (Complex 2). Because subcellular localization has been shown to be important for the function of both NS1 and Crk (Ayllon localization has been shown to be important for the function of both NS1 and Crk (Ayllon and Garcia-Sastre, 2015; Fish et al., 1999; Smith et al., 2002), we also wanted to study and Garcia-Sastre, 2015; Fish et al., 1999; Smith et al., 2002), we also wanted to study the distribution of the Crk-NS1 complex in the host cell. We demonstrated that the SH3 the distribution of the Crk-NS1 complex in the host cell. We demonstrated that the SH3 binding motif containing NS1 proteins were able to induce a robust translocation of Crk binding motif containing NS1 proteins were able to induce a robust translocation of Crk

49 49 proteins from the cytoplasm to the nucleus where the Crk proteins co-localized with NS1 proteins from the cytoplasm to the nucleus where the Crk proteins co-localized with NS1 protein in IAV infected cells. The nuclear localization of Crk proteins could not be protein in IAV infected cells. The nuclear localization of Crk proteins could not be observed when cells were infected with virus that contains an NS1 protein that lacks the observed when cells were infected with virus that contains an NS1 protein that lacks the SH3 binding motif. We further showed that this relocalization of Crk by NS1 did not SH3 binding motif. We further showed that this relocalization of Crk by NS1 did not mediate activation of PI3K/Akt by NS1. Instead, we demonstrated that the translocation mediate activation of PI3K/Akt by NS1. Instead, we demonstrated that the translocation of Crk into nucleus by NS1 protein was associated with a dramatic change in the tyrosine of Crk into nucleus by NS1 protein was associated with a dramatic change in the tyrosine phosphorylation pattern of nuclear proteins. phosphorylation pattern of nuclear proteins. The activation of PI3K/Akt pathway by NS1 protein has been reported to be beneficial The activation of PI3K/Akt pathway by NS1 protein has been reported to be beneficial for the replication and virulence of several IAV strains (Ayllon et al., 2012a; Hale et al., for the replication and virulence of several IAV strains (Ayllon et al., 2012a; Hale et al., 2006; Hrincius et al., 2012; Shin et al., 2007a). In study I we reported that NS1 proteins 2006; Hrincius et al., 2012; Shin et al., 2007a). In study I we reported that NS1 proteins of Spanish Flu and many avian origin IAV strains has evolved with an enhanced capacity of Spanish Flu and many avian origin IAV strains has evolved with an enhanced capacity to activate the PI3K/Akt pathway. This potentiation was shown to be dependent on the to activate the PI3K/Akt pathway. This potentiation was shown to be dependent on the SH3 binding motif found in these proteins. Although the NS1-induced activation of SH3 binding motif found in these proteins. Although the NS1-induced activation of PI3K/Akt signaling is conserved among different IAV strains, the requirement of the PI3K/Akt signaling is conserved among different IAV strains, the requirement of the induction for the virus replication and pathogenesis has been shown to be virus strain induction for the virus replication and pathogenesis has been shown to be virus strain dependent (Ayllon et al., 2012b). Since the SH3 binding motif is highly conserved in dependent (Ayllon et al., 2012b). Since the SH3 binding motif is highly conserved in especially avian IAV strains, and only seldom found in human isolated IAV strains, the especially avian IAV strains, and only seldom found in human isolated IAV strains, the ability to activate the PI3K pathway more effectively must provide the avian viruses with ability to activate the PI3K pathway more effectively must provide the avian viruses with direct replicative advantage in avian host. Even though many human IAV strains require direct replicative advantage in avian host. Even though many human IAV strains require the NS1 induced PI3K activation for efficient virus replication, providing the viruses with the NS1 induced PI3K activation for efficient virus replication, providing the viruses with SH3 binding competent NS1 protein does not have positive (or negative) effect on viral SH3 binding competent NS1 protein does not have positive (or negative) effect on viral replication or virulence (Hale et al., 2010b; Hsiang et al., 2012). However, some replication or virulence (Hale et al., 2010b; Hsiang et al., 2012). However, some exceptions also exist. The human isolated A/PR8 IAV strain was shown to be more exceptions also exist. The human isolated A/PR8 IAV strain was shown to be more pathogenic in mice when the SH3 binding motif was introduced to its NS1 protein pathogenic in mice when the SH3 binding motif was introduced to its NS1 protein (Hrincius et al., 2015). (Hrincius et al., 2015).

5.2 Crk-NS1-PI3K complex formation 5.2 Crk-NS1-PI3K complex formation The NS1-mediated activation of the PI3K pathway has been reported to require a direct The NS1-mediated activation of the PI3K pathway has been reported to require a direct association of NS1 with the p85β regulatory subunit of PI3K (Hale et al., 2006; Shin et association of NS1 with the p85β regulatory subunit of PI3K (Hale et al., 2006; Shin et al., 2007b). The results by Shin et al. indicated that NS1-p85 interaction is mediated by al., 2007b). The results by Shin et al. indicated that NS1-p85 interaction is mediated by the SH3 domain of p85 (Shin et al., 2007a). However, in study I, we were not able to the SH3 domain of p85 (Shin et al., 2007a). However, in study I, we were not able to observe any measurable association between SH3 domains of p85α or p85β and NS1. observe any measurable association between SH3 domains of p85α or p85β and NS1. The proposed SH3 binding motif located around amino acid 164, different from the Crk- The proposed SH3 binding motif located around amino acid 164, different from the Crk- binding site, was later demonstrated to be important for binding to p85 but not through binding site, was later demonstrated to be important for binding to p85 but not through SH3 domain (Hale et al., 2010a). Earlier studies have also linked the Crk proteins to the SH3 domain (Hale et al., 2010a). Earlier studies have also linked the Crk proteins to the activation of PI3K signaling by binding to a proline-rich motif in p85 regulatory subunit activation of PI3K signaling by binding to a proline-rich motif in p85 regulatory subunit of PI3K via their nSH3 domain (Akagi et al., 2002; Akagi et al., 2000; Gelkop et al., 2001; of PI3K via their nSH3 domain (Akagi et al., 2002; Akagi et al., 2000; Gelkop et al., 2001; Sattler et al., 1997). Our results in study II confirmed that Crk proteins are physically Sattler et al., 1997). Our results in study II confirmed that Crk proteins are physically coupled to the activation of PI3K/Akt pathway. We described that the potentiation of coupled to the activation of PI3K/Akt pathway. We described that the potentiation of pathway activation by SH3 binding competent NS1 proteins is caused by their ability to pathway activation by SH3 binding competent NS1 proteins is caused by their ability to recruit Crk adaptor proteins to the PI3K complex more efficiently than the SH3 binding recruit Crk adaptor proteins to the PI3K complex more efficiently than the SH3 binding incompetent NS1 proteins. The SH3 binding motif in NS1 directs the assembly of a novel incompetent NS1 proteins. The SH3 binding motif in NS1 directs the assembly of a novel trimeric complex (p85β-NS1-Crk; Complex 1), where NS1 connects the p85β and Crk. trimeric complex (p85β-NS1-Crk; Complex 1), where NS1 connects the p85β and Crk. Interestingly, we could also demonstrate that Crk proteins are involved in PI3K activation Interestingly, we could also demonstrate that Crk proteins are involved in PI3K activation also by NS1 proteins that lack the SH3 binding site. In this case, another trimeric complex also by NS1 proteins that lack the SH3 binding site. In this case, another trimeric complex of NS1, Crk, and p85β-Crk formed, where instead of NS1, the p85β serves as a bridging of NS1, Crk, and p85β-Crk formed, where instead of NS1, the p85β serves as a bridging factor by binding to both NS1 and Crk (NS1-p85β-Crk; Complex 2). Yet, we could only factor by binding to both NS1 and Crk (NS1-p85β-Crk; Complex 2). Yet, we could only

50 50 observe the Complex 2 when p85β was overexpressed, which led us to speculate that observe the Complex 2 when p85β was overexpressed, which led us to speculate that the weaker activation of PI3K/Akt observed by SH3 binding incompetent NS1 proteins the weaker activation of PI3K/Akt observed by SH3 binding incompetent NS1 proteins might be because of the low cellular levels of p85β and lower affinity of p85β to the Crk might be because of the low cellular levels of p85β and lower affinity of p85β to the Crk nSH3 domain. Indeed, we could demonstrate that overexpression of Crk proteins could nSH3 domain. Indeed, we could demonstrate that overexpression of Crk proteins could compensate the lower PI3K/Akt induction seen by the SH3 binding incompetent NS1 compensate the lower PI3K/Akt induction seen by the SH3 binding incompetent NS1 proteins compared to SH3 binding competent NS1. Thus, indicating that the SH3 binding proteins compared to SH3 binding competent NS1. Thus, indicating that the SH3 binding competent NS1 provides more efficient recruitment of Crk to the PI3K leading to the competent NS1 provides more efficient recruitment of Crk to the PI3K leading to the formation of Complex 1 and enhanced activation of the pathway. However, the NS1- formation of Complex 1 and enhanced activation of the pathway. However, the NS1- mediated PI3K/Akt activation was seen to be solely depended on the interaction of NS1 mediated PI3K/Akt activation was seen to be solely depended on the interaction of NS1 with p85β, which was in line with earlier studies by others (Hale et al., 2006; Shin et al., with p85β, which was in line with earlier studies by others (Hale et al., 2006; Shin et al., 2007b), and the association of NS1 with Crk-SH3 domain was able to only enhance the 2007b), and the association of NS1 with Crk-SH3 domain was able to only enhance the capacity of NS1 to induce the pathway. capacity of NS1 to induce the pathway. Structural studies on NS1-PI3K complex have revealed the mechanism of NS1-induced Structural studies on NS1-PI3K complex have revealed the mechanism of NS1-induced activation of PI3K, but the precise role of Crk proteins in this requires further studies. activation of PI3K, but the precise role of Crk proteins in this requires further studies. Binding of NS1 to the p85β breaks up the inhibitory contacts between the p110 and Binding of NS1 to the p85β breaks up the inhibitory contacts between the p110 and p85β (Hale et al., 2010a). A possible scenario is that Crk proteins or some Crk associated p85β (Hale et al., 2010a). A possible scenario is that Crk proteins or some Crk associated protein directly contributes to the activation of the complex or is involved in dissociation protein directly contributes to the activation of the complex or is involved in dissociation of the negative regulation between p110 and p85β. Studies by others have reported that of the negative regulation between p110 and p85β. Studies by others have reported that the Crk SH2 domain mediates the activation of PI3K by binding to Cbl and activating the the Crk SH2 domain mediates the activation of PI3K by binding to Cbl and activating the Src kinases (Akagi et al., 2002; Song et al., 2010). Thus, the role of Crk in NS1-induced Src kinases (Akagi et al., 2002; Song et al., 2010). Thus, the role of Crk in NS1-induced PI3K signaling could be to recruit Src kinases to the NS1-PI3K complex through binding PI3K signaling could be to recruit Src kinases to the NS1-PI3K complex through binding to the Cbl. Since several alternative p85 and p110 isoforms contribute to the formation to the Cbl. Since several alternative p85 and p110 isoforms contribute to the formation of PI3K signaling complex depending on the initial stimulus (Vanhaesebroeck et al., of PI3K signaling complex depending on the initial stimulus (Vanhaesebroeck et al., 2010), another possibility for the role of Crk in NS1-mediated PI3K activation could be 2010), another possibility for the role of Crk in NS1-mediated PI3K activation could be that Crk anchors the PI3K-NS1 complex to a subcellular compartment that is beneficial that Crk anchors the PI3K-NS1 complex to a subcellular compartment that is beneficial for the pathway activation by NS1. Yet, we could only observe the Complex 2 when p85β for the pathway activation by NS1. Yet, we could only observe the Complex 2 when p85β was overexpressed, which led us to speculate that the weaker activation of PI3K/Akt was overexpressed, which led us to speculate that the weaker activation of PI3K/Akt observed by SH3 binding incompetent NS1 proteins might be because of the low cellular observed by SH3 binding incompetent NS1 proteins might be because of the low cellular levels of p85β and lower affinity of p85β to the Crk nSH3 domain. Indeed, we could levels of p85β and lower affinity of p85β to the Crk nSH3 domain. Indeed, we could demonstrate that overexpression of Crk proteins could compensate the lower PI3K/Akt demonstrate that overexpression of Crk proteins could compensate the lower PI3K/Akt induction seen by the SH3 binding incompetent NS1 proteins compared to SH3 binding induction seen by the SH3 binding incompetent NS1 proteins compared to SH3 binding competent NS1. Thus, indicating that the SH3 binding competent NS1 provides more competent NS1. Thus, indicating that the SH3 binding competent NS1 provides more efficient recruitment of Crk to the PI3K leading to the formation of Complex 1 and efficient recruitment of Crk to the PI3K leading to the formation of Complex 1 and enhanced activation of the pathway. However, the NS1-mediated PI3K/Akt activation enhanced activation of the pathway. However, the NS1-mediated PI3K/Akt activation was seen to be solely depended on the interaction of NS1 with p85β, which was in line was seen to be solely depended on the interaction of NS1 with p85β, which was in line with earlier studies by others (Hale et al., 2006; Shin et al., 2007b), and the association with earlier studies by others (Hale et al., 2006; Shin et al., 2007b), and the association of NS1 with Crk-SH3 domain was able to only potentiate the capacity of NS1 to induce of NS1 with Crk-SH3 domain was able to only potentiate the capacity of NS1 to induce the pathway. the pathway.

5.3 Nuclear localization of Crk-NS1 complex 5.3 Nuclear localization of Crk-NS1 complex Since the replication of IAV occurs in the nucleus of the host cell, manipulation of nuclear Since the replication of IAV occurs in the nucleus of the host cell, manipulation of nuclear environment during the infection is important for promoting virus propagation. Both environment during the infection is important for promoting virus propagation. Both NS1 and Crk proteins have described to carry out distinct functions in the nucleus. The NS1 and Crk proteins have described to carry out distinct functions in the nucleus. The NLS sequences efficiently target the NS1 protein into the nucleus, where it, for example, NLS sequences efficiently target the NS1 protein into the nucleus, where it, for example, inhibits host gene expression by binding to CPSF30. Crk proteins have also been inhibits host gene expression by binding to CPSF30. Crk proteins have also been described with different nuclear functions but since they lack any NLS, their transport described with different nuclear functions but since they lack any NLS, their transport from the cytoplasm to the nucleus is regulated by their interaction partners. In study III, from the cytoplasm to the nucleus is regulated by their interaction partners. In study III,

51 51 we reported a robust nuclear translocation of Crk proteins by SH3 binding competent we reported a robust nuclear translocation of Crk proteins by SH3 binding competent NS1 which was further associated with tyrosine phosphorylation of an approximately NS1 which was further associated with tyrosine phosphorylation of an approximately 135 kDa nuclear protein. The size of the protein correlates with the cytoplasmic tyrosine 135 kDa nuclear protein. The size of the protein correlates with the cytoplasmic tyrosine phosphorylated p130Cas, an adaptor protein that binds to the Crk SH2-domain (Birge et phosphorylated p130Cas, an adaptor protein that binds to the Crk SH2-domain (Birge et al., 1992; Sakai et al., 1994). Another tyrosine phosphorylated Crk-interaction partner al., 1992; Sakai et al., 1994). Another tyrosine phosphorylated Crk-interaction partner that also correlates with the size of the unknown tyrosine phosphorylated protein in the that also correlates with the size of the unknown tyrosine phosphorylated protein in the nuclear fraction is c-Abl. Crk associates with c-Abl through the nSH3 domain and the nuclear fraction is c-Abl. Crk associates with c-Abl through the nSH3 domain and the interaction causes the autophosphorylation and activation of c-Abl (Brasher and Van interaction causes the autophosphorylation and activation of c-Abl (Brasher and Van Etten, 2000; Shishido et al., 2001). So far we have not been able to show that either one Etten, 2000; Shishido et al., 2001). So far we have not been able to show that either one of these proteins would be a part of Crk-NS1 complex in the nucleus. Since the of these proteins would be a part of Crk-NS1 complex in the nucleus. Since the association of Crk with c-Abl is mediated by the nSH3 domain, the Crk-NS1 interaction association of Crk with c-Abl is mediated by the nSH3 domain, the Crk-NS1 interaction would have to be disrupted before the Crk-c-Abl interaction could happen. Additionally, would have to be disrupted before the Crk-c-Abl interaction could happen. Additionally, the association of c-Abl with Crk could happen indirectly since NS1 has also been the association of c-Abl with Crk could happen indirectly since NS1 has also been reported to interact with c-Abl (Hrincius et al., 2014). Although this remains an reported to interact with c-Abl (Hrincius et al., 2014). Although this remains an interesting scenario, the interaction of NS1 with c-Abl was linked to inhibition of the c- interesting scenario, the interaction of NS1 with c-Abl was linked to inhibition of the c- Abl, not activation. The tyrosine phosphorylated protein observed in the nuclear fraction Abl, not activation. The tyrosine phosphorylated protein observed in the nuclear fraction could also be a protein that is not physically linked to Crk. As the Crk proteins are could also be a protein that is not physically linked to Crk. As the Crk proteins are involved in the regulation of a large number of different protein kinases both in the involved in the regulation of a large number of different protein kinases both in the cytoplasm and the nucleus, the translocation of Crk into nucleus by NS1 may initiate a cytoplasm and the nucleus, the translocation of Crk into nucleus by NS1 may initiate a signaling cascade of different kinases and the tyrosine phosphorylated protein we signaling cascade of different kinases and the tyrosine phosphorylated protein we observe could be acting somewhere downstream of Crk. Further work is needed to observe could be acting somewhere downstream of Crk. Further work is needed to reveal the identity of the tyrosine phosphorylated protein and its role in IAV infection. reveal the identity of the tyrosine phosphorylated protein and its role in IAV infection. Previous studies have described different nuclear functions for Crk proteins. It has been Previous studies have described different nuclear functions for Crk proteins. It has been shown that in response to type I IFNs and various cytokines, CrkL binds to shown that in response to type I IFNs and various cytokines, CrkL binds to phosphorylated Stat5 via the CrkL SH2 domain (Fish et al., 1999; Grumbach et al., 2001; phosphorylated Stat5 via the CrkL SH2 domain (Fish et al., 1999; Grumbach et al., 2001; Lekmine et al., 2002; Rhodes et al., 2000; Uddin et al., 2003). The resulting CrkL-Stat5 Lekmine et al., 2002; Rhodes et al., 2000; Uddin et al., 2003). The resulting CrkL-Stat5 complex translocates into the nucleus where it binds to Stat5-responsive elements to complex translocates into the nucleus where it binds to Stat5-responsive elements to induce the expression of IFN stimulated genes. Thus, the NS1-mediated translocation of induce the expression of IFN stimulated genes. Thus, the NS1-mediated translocation of CrkL to the nucleus we reported in study III could provide another layer of the inhibition CrkL to the nucleus we reported in study III could provide another layer of the inhibition of IFN stimulated genes already described for NS1 protein. Importantly, transportation of IFN stimulated genes already described for NS1 protein. Importantly, transportation of CrkII into the nucleus has been reported to be pro-apoptotic by activating caspases of CrkII into the nucleus has been reported to be pro-apoptotic by activating caspases and binding to the cell cycle regulator Wee1 through the CrkII SH2 domain (Smith et al., and binding to the cell cycle regulator Wee1 through the CrkII SH2 domain (Smith et al., 2000; Smith et al., 2002). Apoptosis and the activation of caspases is important for 2000; Smith et al., 2002). Apoptosis and the activation of caspases is important for efficient nuclear exit of IAV vRNP complexes (Muhlbauer et al., 2015; Wurzer et al., efficient nuclear exit of IAV vRNP complexes (Muhlbauer et al., 2015; Wurzer et al., 2003). Thus, another interesting scenario for NS1-mediated Crk nuclear translocation 2003). Thus, another interesting scenario for NS1-mediated Crk nuclear translocation would be to induce apoptosis in order to promote the exit of vRNP from the nucleus. would be to induce apoptosis in order to promote the exit of vRNP from the nucleus. Underneath the nuclear envelope lies a protein meshwork called the nuclear lamina, Underneath the nuclear envelope lies a protein meshwork called the nuclear lamina, composed of nuclear lamins, which is thought to be a common target especially for composed of nuclear lamins, which is thought to be a common target especially for nuclear replicating viruses. Our unpublished data indicates that viruses expressing SH3 nuclear replicating viruses. Our unpublished data indicates that viruses expressing SH3 binding competent NS1 may induce the cleavage and dispersion of nuclear lamins binding competent NS1 may induce the cleavage and dispersion of nuclear lamins compared to viruses in which NS1 lacks the Crk binding capacity. Targeting lamins could compared to viruses in which NS1 lacks the Crk binding capacity. Targeting lamins could be a possible mechanism for viruses bearing Crk binding competent NS1 to promote viral be a possible mechanism for viruses bearing Crk binding competent NS1 to promote viral replication. replication.

5.4 Multiple functions of Crk-NS1 interaction 5.4 Multiple functions of Crk-NS1 interaction Crk proteins regulate a large set of cellular signaling events. Similarly, NS1 has multiple Crk proteins regulate a large set of cellular signaling events. Similarly, NS1 has multiple functions and numerous interaction partners in the host cell. Considering this, it is not a functions and numerous interaction partners in the host cell. Considering this, it is not a

52 52 surprise that Crk-NS1 interaction has been reported to be involved in several cell surprise that Crk-NS1 interaction has been reported to be involved in several cell functions as well. In addition to our observations that Crk-NS1 interaction is involved in functions as well. In addition to our observations that Crk-NS1 interaction is involved in the superactivation of PI3K and in manipulating the intracellular distribution of Crk the superactivation of PI3K and in manipulating the intracellular distribution of Crk proteins, which leads to changes in tyrosine phosphorylation of nuclear proteins, others proteins, which leads to changes in tyrosine phosphorylation of nuclear proteins, others have noticed additional functions for this interaction, namely the suppression of c-Abl have noticed additional functions for this interaction, namely the suppression of c-Abl and JNK signaling (Hrincius et al., 2014; Hrincius et al., 2010). How is the Crk-NS1 and JNK signaling (Hrincius et al., 2014; Hrincius et al., 2010). How is the Crk-NS1 complex able to conduct all these functions in infected cell? NS1 protein is expressed in complex able to conduct all these functions in infected cell? NS1 protein is expressed in considerable amounts in infected cells and the expression level of Crk proteins is also considerable amounts in infected cells and the expression level of Crk proteins is also high. Thus, Crk-NS1 complex would probably be able to participate in different signaling high. Thus, Crk-NS1 complex would probably be able to participate in different signaling routes at the same time and either one of the proteins could be the limiting factor. NS1 routes at the same time and either one of the proteins could be the limiting factor. NS1 protein has been speculated to be temporally regulated in order to execute all its protein has been speculated to be temporally regulated in order to execute all its different tasks at the right time, thus all the distinct functions described for Crk-NS1 different tasks at the right time, thus all the distinct functions described for Crk-NS1 complex could also be coordinated to happen in different time points of infection. In complex could also be coordinated to happen in different time points of infection. In addition, one has to bear in mind the differences in the signaling characteristics of the addition, one has to bear in mind the differences in the signaling characteristics of the three Crk family members. Hrincius et al. reported that CrkI was the main family member three Crk family members. Hrincius et al. reported that CrkI was the main family member participating in the inhibition of JNK pathway and apoptosis (Hrincius et al., 2010). We participating in the inhibition of JNK pathway and apoptosis (Hrincius et al., 2010). We were not able to see any variation between the different Crk proteins in the ability to were not able to see any variation between the different Crk proteins in the ability to enhance the NS1-induced PI3K activation in study II. When we studied the localization enhance the NS1-induced PI3K activation in study II. When we studied the localization of the Crk-NS1 complex in study III, we could observe more pronounced translocation of the Crk-NS1 complex in study III, we could observe more pronounced translocation of CrkL than CrkI or CrkII into nucleus, but the possible differences of these proteins for of CrkL than CrkI or CrkII into nucleus, but the possible differences of these proteins for the tyrosine phosphorylation of the nuclear protein observed or other possible nuclear the tyrosine phosphorylation of the nuclear protein observed or other possible nuclear functions remain to be studied. functions remain to be studied.

5.5 Diversity in the SH3 binding motif of NS1 5.5 Diversity in the SH3 binding motif of NS1 The NS1 sequence at the SH3 binding motif (amino acids 212-217) is diverse among The NS1 sequence at the SH3 binding motif (amino acids 212-217) is diverse among different IAV strains. While most avian IAV NS1 proteins contain the Crk-SH3 binding different IAV strains. While most avian IAV NS1 proteins contain the Crk-SH3 binding motif, only few human IAV NS1 proteins contain the motif. The critical amino acids, that motif, only few human IAV NS1 proteins contain the motif. The critical amino acids, that we reported to be important for the SH3-mediated binding of NS1 to Crk in study I (P212, we reported to be important for the SH3-mediated binding of NS1 to Crk in study I (P212, P215, and K217) provides the major variation sites observed for NS1 proteins of different P215, and K217) provides the major variation sites observed for NS1 proteins of different IAV strains. Sequence alignment of multiple NS1 proteins from avian IAV isolates until IAV strains. Sequence alignment of multiple NS1 proteins from avian IAV isolates until this date reveals a consensus sequence of 212PPLPPK217 (NCBI, 2016), showing that the this date reveals a consensus sequence of 212PPLPPK217 (NCBI, 2016), showing that the SH3 binding site is highly conserved in NS1 proteins avian IAVs. In contrast, sequence SH3 binding site is highly conserved in NS1 proteins avian IAVs. In contrast, sequence alignment of human IAV isolate NS1 proteins reveals that these NS1 proteins contain a alignment of human IAV isolate NS1 proteins reveals that these NS1 proteins contain a consensus sequence of 212PPLTPK217 (virus isolates from years 2009-2016). Thus, most consensus sequence of 212PPLTPK217 (virus isolates from years 2009-2016). Thus, most of the avian influenza A viruses have a proline at amino acid 215, whereas human IAV of the avian influenza A viruses have a proline at amino acid 215, whereas human IAV strains contain a threonine in this position but variations at different positions also strains contain a threonine in this position but variations at different positions also occur. Avian IAV subtype H5N1 offers an exception to this rule, since the consensus for occur. Avian IAV subtype H5N1 offers an exception to this rule, since the consensus for NS1 in these viruses is 212LPLPPN217 (virus isolates from years 2009-2016), where two NS1 in these viruses is 212LPLPPN217 (virus isolates from years 2009-2016), where two amino acids important for the binding have been substituted with other residues (P212L amino acids important for the binding have been substituted with other residues (P212L and K217N). Interestingly, the NS1 protein of famous human pandemic IAV strain, the and K217N). Interestingly, the NS1 protein of famous human pandemic IAV strain, the 1918 Spanish Flu, possessed the SH3 domain binding site, but the motif was lost during 1918 Spanish Flu, possessed the SH3 domain binding site, but the motif was lost during further adaptation of the virus to human. further adaptation of the virus to human.

5.6 Significance of the NS1 SH3 binding motif for IAV 5.6 Significance of the NS1 SH3 binding motif for IAV The NS1 gene of most seasonal IAV strains would need just a single nucleotide change The NS1 gene of most seasonal IAV strains would need just a single nucleotide change to provide the NS1 protein with the capacity to bind Crk SH3 domain. If it would be to provide the NS1 protein with the capacity to bind Crk SH3 domain. If it would be beneficial for viral replication and virulence, it could be expected to happen quickly. beneficial for viral replication and virulence, it could be expected to happen quickly.

53 53 Instead, the requirement of the Crk-binding motif in NS1 protein for IAV replication and Instead, the requirement of the Crk-binding motif in NS1 protein for IAV replication and pathogenesis seems to be virus strain specific. For example, a single nucleotide change pathogenesis seems to be virus strain specific. For example, a single nucleotide change in the NS1 sequence of the 2009 pandemic Swine Flu (A/California/04/09) created a in the NS1 sequence of the 2009 pandemic Swine Flu (A/California/04/09) created a functional Crk-binding motif (E217K), but did not enhance virus replication in human or functional Crk-binding motif (E217K), but did not enhance virus replication in human or swine cells (Hale et al., 2010b). The virulence or the transmission of the virus was also swine cells (Hale et al., 2010b). The virulence or the transmission of the virus was also not affected. Likewise, mutation of T215 to proline to create an SH3 binding site in not affected. Likewise, mutation of T215 to proline to create an SH3 binding site in human IAVs of A/Udorn and the Swine Flu strains did not affect virus replication (Hsiang human IAVs of A/Udorn and the Swine Flu strains did not affect virus replication (Hsiang et al., 2012). Similarly the avian H5N1 subtype IAV, in which NS1 naturally lacks the Crk- et al., 2012). Similarly the avian H5N1 subtype IAV, in which NS1 naturally lacks the Crk- binding site, did not benefit from the introduction of the SH3 binding motif into its NS1 binding site, did not benefit from the introduction of the SH3 binding motif into its NS1 protein (Hrincius et al., 2014). In contrast, avian IAV strain that expresses SH3 binding protein (Hrincius et al., 2014). In contrast, avian IAV strain that expresses SH3 binding competent NS1 replicated less efficiently when the motif was mutated. On the other competent NS1 replicated less efficiently when the motif was mutated. On the other hand, human IAV virus, A/PR8 was more pathogenic in mice, when SH3 binding site was hand, human IAV virus, A/PR8 was more pathogenic in mice, when SH3 binding site was introduced in NS1 (Hrincius et al., 2015). Thus, the requirement for the Crk binding site introduced in NS1 (Hrincius et al., 2015). Thus, the requirement for the Crk binding site in NS1 is strikingly virus strain dependent, and the viruses naturally lacking the site do in NS1 is strikingly virus strain dependent, and the viruses naturally lacking the site do not (with some notable exceptions) benefit from the addition of this site. The reason not (with some notable exceptions) benefit from the addition of this site. The reason why most avian IAVs seem to benefit from the ability to modulate Crk signaling in the why most avian IAVs seem to benefit from the ability to modulate Crk signaling in the host cell and why it is lost when the virus is transmitted to human host may lie in the host cell and why it is lost when the virus is transmitted to human host may lie in the natural environment these viruses encounter. The major replication site for avian IAVs natural environment these viruses encounter. The major replication site for avian IAVs in their natural host is the intestine tract, whereas human IAVs infect mainly the trachea. in their natural host is the intestine tract, whereas human IAVs infect mainly the trachea. The differences in protein expression and in the temperature of the environment may The differences in protein expression and in the temperature of the environment may dictate the necessity of the Crk binding property of NS1 protein for the virus. The Crk dictate the necessity of the Crk binding property of NS1 protein for the virus. The Crk proteins are ubiquitously expressed in different human tissues and they are well proteins are ubiquitously expressed in different human tissues and they are well conserved between different species. However, the expression levels of these proteins conserved between different species. However, the expression levels of these proteins in the avian tissues is not known and should be studied. Remarkably, in study I we in the avian tissues is not known and should be studied. Remarkably, in study I we showed that the NS1 protein of the IAV strain that caused the most severe pandemic showed that the NS1 protein of the IAV strain that caused the most severe pandemic known of all time, the highly pathogenic Spanish Flu, contain a functional SH3 binding known of all time, the highly pathogenic Spanish Flu, contain a functional SH3 binding motif. The Spanish Flu IAV has been reported to be extraordinarily virulent and possess motif. The Spanish Flu IAV has been reported to be extraordinarily virulent and possess higher replication capacity in cell culture and mice than the reference IAVs (Kash et al., higher replication capacity in cell culture and mice than the reference IAVs (Kash et al., 2006; Tumpey et al., 2005). Moreover, the Spanish Flu NS1 has been reported to be 2006; Tumpey et al., 2005). Moreover, the Spanish Flu NS1 has been reported to be more potent in regulating gene expression in host cell compared to NS1 proteins of more potent in regulating gene expression in host cell compared to NS1 proteins of other IAV strains (Geiss et al., 2002). The SH3 binding site that provides the Spanish Flu other IAV strains (Geiss et al., 2002). The SH3 binding site that provides the Spanish Flu NS1 protein with capacity to exploit the host cell signaling machinery by binding to Crk NS1 protein with capacity to exploit the host cell signaling machinery by binding to Crk proteins may have contributed to the very highly pathogenic nature the virus had. The proteins may have contributed to the very highly pathogenic nature the virus had. The adaptation of the virus to contain the SH3 binding site might provide the virus with adaptation of the virus to contain the SH3 binding site might provide the virus with increased replicative advantage or pathogenic potential also in human cells, which increased replicative advantage or pathogenic potential also in human cells, which provides a potential threat in novel zoonotic pandemic viruses. provides a potential threat in novel zoonotic pandemic viruses.

54 54 6 CONCLUSIONS 6 CONCLUSIONS

In this thesis, we found a novel, functional SH3 binding motif in the NS1 protein of In this thesis, we found a novel, functional SH3 binding motif in the NS1 protein of Spanish Flu and most avian isolated IAV strains but not in NS1 proteins of most human Spanish Flu and most avian isolated IAV strains but not in NS1 proteins of most human IAV strains. The site was shown to mediate interaction with the nSH3 domain of the Crk- IAV strains. The site was shown to mediate interaction with the nSH3 domain of the Crk- family adaptor proteins. This association provides NS1 protein with enhanced capacity family adaptor proteins. This association provides NS1 protein with enhanced capacity to induce PI3K/Akt signaling. We also described the molecular mechanism behind the to induce PI3K/Akt signaling. We also described the molecular mechanism behind the PI3K superactivation by SH3 binding competent NS1 proteins. The potentiation of the PI3K superactivation by SH3 binding competent NS1 proteins. The potentiation of the pathway was shown to be a result of a reorganization of the natural PI3K-Crk complex pathway was shown to be a result of a reorganization of the natural PI3K-Crk complex and the formation of a novel trimeric complex of PI3K, NS1 and Crk. Moreover, we found and the formation of a novel trimeric complex of PI3K, NS1 and Crk. Moreover, we found that Crk proteins also have a general role in NS1-mediated activation of PI3K that is that Crk proteins also have a general role in NS1-mediated activation of PI3K that is independent from the SH3 binding capacity of NS1. In addition, we described another independent from the SH3 binding capacity of NS1. In addition, we described another function for Crk-NS1 interaction. The binding of Crk to NS1 was shown to lead to a robust function for Crk-NS1 interaction. The binding of Crk to NS1 was shown to lead to a robust translocation of cytoplasmic Crk proteins into the nucleus. This function was translocation of cytoplasmic Crk proteins into the nucleus. This function was independent from the induction of the PI3K pathway. Instead, the potent translocation independent from the induction of the PI3K pathway. Instead, the potent translocation of Crk proteins into the nucleus was linked to tyrosine phosphorylation of a nuclear of Crk proteins into the nucleus was linked to tyrosine phosphorylation of a nuclear protein. protein. The results presented here describe two new mechanisms for IAV to regulate host cell The results presented here describe two new mechanisms for IAV to regulate host cell signaling by exploiting host cell SH3 domains. While the capability to activate PI3K signaling by exploiting host cell SH3 domains. While the capability to activate PI3K signaling by NS1 protein is conserved among most IAV strains, our studies revealed that signaling by NS1 protein is conserved among most IAV strains, our studies revealed that the SH3 binding competent NS1 proteins are exceptionally potent PI3K activators. In the SH3 binding competent NS1 proteins are exceptionally potent PI3K activators. In addition, the SH3 binding site enables the NS1 proteins to drag the Crk proteins into the addition, the SH3 binding site enables the NS1 proteins to drag the Crk proteins into the nucleus and consequently manipulate the nuclear environment. Importantly, the nucleus and consequently manipulate the nuclear environment. Importantly, the presented data also reveal that Crk proteins are important general host cell co-factors presented data also reveal that Crk proteins are important general host cell co-factors of IAV infection, independent from the NS1 Crk SH3 binding motif. The fact that some of IAV infection, independent from the NS1 Crk SH3 binding motif. The fact that some IAV strains have evolved a more efficient strategy to take maximal use of the Crk IAV strains have evolved a more efficient strategy to take maximal use of the Crk proteins to regulate host cell signaling in the cytoplasm and nucleus emphasizes the proteins to regulate host cell signaling in the cytoplasm and nucleus emphasizes the importance of these proteins as NS1-interactor partners in IAV infection. importance of these proteins as NS1-interactor partners in IAV infection. Further studies on this subject will provide deeper knowledge on the cell biology of IAV Further studies on this subject will provide deeper knowledge on the cell biology of IAV replication. They will provide a new perspective to the IAV strain-specific variation of replication. They will provide a new perspective to the IAV strain-specific variation of the SH3-binding capacity of NS1, and hopefully open new avenues for anti-IAV the SH3-binding capacity of NS1, and hopefully open new avenues for anti-IAV therapeutic development. It would be important to study the relevance of the NS1 SH3 therapeutic development. It would be important to study the relevance of the NS1 SH3 binding motif in avian cells. Detailed characterization of the changes in the nuclear binding motif in avian cells. Detailed characterization of the changes in the nuclear protein phosphorylation pattern is also clearly needed in order to understand better the protein phosphorylation pattern is also clearly needed in order to understand better the importance of the relocalization of Crk proteins from the cytoplasm to the nucleus by importance of the relocalization of Crk proteins from the cytoplasm to the nucleus by NS1 for IAV replication and pathogenesis. The role of NS1-Crk interaction for IAV cell NS1 for IAV replication and pathogenesis. The role of NS1-Crk interaction for IAV cell biology is undoubtedly important, and deserves special attention also in the future. biology is undoubtedly important, and deserves special attention also in the future.

55 55 7 ACKNOWLEDGEMENTS 7 ACKNOWLEDGEMENTS

This study was carried out at the Department of Virology, University of Helsinki. I want This study was carried out at the Department of Virology, University of Helsinki. I want to thank the head of the department, Professor Kalle Saksela for providing excellent and to thank the head of the department, Professor Kalle Saksela for providing excellent and welcoming working facilities. welcoming working facilities. I am deeply grateful to my supervisor, Professor Kalle Saksela, for his outstanding I am deeply grateful to my supervisor, Professor Kalle Saksela, for his outstanding guidance, support and patience during my thesis research. You were always able to give guidance, support and patience during my thesis research. You were always able to give me new confidence in my project when never I was in doubt. Thank you for giving me me new confidence in my project when never I was in doubt. Thank you for giving me this opportunity. this opportunity. I warmly thank Docent Tero Ahola and Docent Denis Kainov for reviewing my thesis in I warmly thank Docent Tero Ahola and Docent Denis Kainov for reviewing my thesis in very short period of time and for providing valuable comments that improved my thesis very short period of time and for providing valuable comments that improved my thesis significantly. Professor Ilkka Julkunen and Professor Alexander Plyusnin are thanked for significantly. Professor Ilkka Julkunen and Professor Alexander Plyusnin are thanked for participating in my thesis committee. I also want take this opportunity to thank Docent participating in my thesis committee. I also want take this opportunity to thank Docent Varpu Marjomäki for accepting the role of the opponent in my thesis dissertation. Varpu Marjomäki for accepting the role of the opponent in my thesis dissertation. All the co-authors who contributed to my work are also thanked. Constanze Schmotz, All the co-authors who contributed to my work are also thanked. Constanze Schmotz, Arunas Kazlauskas, Erkko Ylösmäki, Riku Fagerlund, Inka Kuisma, Ilkka Julkunen, Krister Arunas Kazlauskas, Erkko Ylösmäki, Riku Fagerlund, Inka Kuisma, Ilkka Julkunen, Krister Melén, and Thedi Ziegler are the persons without whom this thesis could not have been Melén, and Thedi Ziegler are the persons without whom this thesis could not have been possible. possible. This thesis has been partly financed by Doctoral Programme in Biomedicine, University This thesis has been partly financed by Doctoral Programme in Biomedicine, University of Helsinki. Suomen Tiedeseura, Waldemar von Frenckells stiftelse, and Medicinska of Helsinki. Suomen Tiedeseura, Waldemar von Frenckells stiftelse, and Medicinska Understödsföreningen Liv och Hälsa are also thanked for financial support during the Understödsföreningen Liv och Hälsa are also thanked for financial support during the completion of this work. completion of this work.

My warmest gratitude goes to all the current and former members of the Saksela lab. My warmest gratitude goes to all the current and former members of the Saksela lab. Kalle, Tapio, Virpi, Constanze, Hannamari, Annika, Silja, Iivari, Erkko, Riku, Zhao, Kalle, Tapio, Virpi, Constanze, Hannamari, Annika, Silja, Iivari, Erkko, Riku, Zhao, Subhash, Inka, Kristina, Tina, Matjaz, Arunas, Jacob, Yoke, Sergio, Hyunseok, Jubauer, Subhash, Inka, Kristina, Tina, Matjaz, Arunas, Jacob, Yoke, Sergio, Hyunseok, Jubauer, Marcal, Arnab, Johanna, Misao, Anette, Timo, and Virpi K are all thanked for creating a Marcal, Arnab, Johanna, Misao, Anette, Timo, and Virpi K are all thanked for creating a friendly and pleasant working atmosphere in the lab, not forgetting all the scientific help friendly and pleasant working atmosphere in the lab, not forgetting all the scientific help you have provided me throughout these years. Especially I would like to thank Tapio for you have provided me throughout these years. Especially I would like to thank Tapio for not just guiding me in the lab and providing invaluable comments and new ideas of my not just guiding me in the lab and providing invaluable comments and new ideas of my work, but also for all the off-topic discussions. I deeply thank Constanze for all the hands- work, but also for all the off-topic discussions. I deeply thank Constanze for all the hands- on help in the lab and for being a true friend for me all these years. I can never forget on help in the lab and for being a true friend for me all these years. I can never forget Jacob and Arunas who were involved in many fun stuff some years ago. And Virpi, thank Jacob and Arunas who were involved in many fun stuff some years ago. And Virpi, thank you for keeping us all in strict order in the lab as well as for all the conversations we have you for keeping us all in strict order in the lab as well as for all the conversations we have had. had.

My deepest gratitude goes to my family. Thank you mom and dad for always supporting My deepest gratitude goes to my family. Thank you mom and dad for always supporting and helping me in all aspects of life. Kiitos! Thank you to my sisters, Eeva and Elina, just and helping me in all aspects of life. Kiitos! Thank you to my sisters, Eeva and Elina, just for being my sisters. I am very happy to have you in my life! I would also like to express for being my sisters. I am very happy to have you in my life! I would also like to express my sincere gratitude to my parents-in-law, Helena and Sauli, for all their support and my sincere gratitude to my parents-in-law, Helena and Sauli, for all their support and help they have provided. I am very grateful to my husband Erkko. Thank you for being help they have provided. I am very grateful to my husband Erkko. Thank you for being there all these years and for pushing me towards this goal. I wouldn’t be here now there all these years and for pushing me towards this goal. I wouldn’t be here now without you! Thank you also for sharing the life and family with me, not forgetting the without you! Thank you also for sharing the life and family with me, not forgetting the

56 56 science part as well. I don’t have enough words to express how grateful I am of my science part as well. I don’t have enough words to express how grateful I am of my children, the jewels of my life. Thank you Emil, Aura and Elsa for being the most honest children, the jewels of my life. Thank you Emil, Aura and Elsa for being the most honest and funniest persons in my life. Don’t ever lose the happiness inside you. and funniest persons in my life. Don’t ever lose the happiness inside you.

Espoo, June 2016 Espoo, June 2016

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Akagi, T., Shishido, T., Murata, K., Hanafusa, H., 2000. v-Crk activates the phosphoinositide 3- Akagi, T., Shishido, T., Murata, K., Hanafusa, H., 2000. v-Crk activates the phosphoinositide 3- kinase/AKT pathway in transformation. Proc Natl Acad Sci U S A 97, 7290-7295. kinase/AKT pathway in transformation. Proc Natl Acad Sci U S A 97, 7290-7295.

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 9, pp. 5719–5727, February 29, 2008 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 9, pp. 5719–5727, February 29, 2008 © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Avian and 1918 Spanish Influenza A Virus NS1 Proteins Bind Avian and 1918 Spanish Influenza A Virus NS1 Proteins Bind to Crk/CrkL Src Homology 3 Domains to Activate Host to Crk/CrkL Src Homology 3 Domains to Activate Host Cell Signaling* Cell Signaling* Received for publication, August 28, 2007, and in revised form, December 27, 2007 Published, JBC Papers in Press, December 28, 2007, DOI 10.1074/jbc.M707195200 Received for publication, August 28, 2007, and in revised form, December 27, 2007 Published, JBC Papers in Press, December 28, 2007, DOI 10.1074/jbc.M707195200 Leena S. Heikkinen‡1, Arunas Kazlauskas‡, Krister Mele´n§, Ralf Wagner¶, Thedi Ziegler§, Ilkka Julkunen§, Leena S. Heikkinen‡1, Arunas Kazlauskas‡, Krister Mele´n§, Ralf Wagner¶, Thedi Ziegler§, Ilkka Julkunen§, and Kalle Saksela‡2 and Kalle Saksela‡2 From the ‡Department of Virology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, From the ‡Department of Virology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Haartmaninkatu 3 (POB 21), FIN-00014, Helsinki, Finland, the §Department of Viral Diseases and Immunology, Haartmaninkatu 3 (POB 21), FIN-00014, Helsinki, Finland, the §Department of Viral Diseases and Immunology, National Public Health Institute, FIN-00300, Helsinki, Finland, and ¶Institute of Medical Microbiology and Hygiene, National Public Health Institute, FIN-00300, Helsinki, Finland, and ¶Institute of Medical Microbiology and Hygiene, University of Regensburg, Franz-Josef-Strausse Allee 11, Regensburg D-93053, Germany University of Regensburg, Franz-Josef-Strausse Allee 11, Regensburg D-93053, Germany

NS1 (nonstructural protein 1) is an important virulence factor tified viral proteins (2). NS1 (nonstructural protein 1) is NS1 (nonstructural protein 1) is an important virulence factor tified viral proteins (2). NS1 (nonstructural protein 1) is of the influenza A virus. We observed that NS1 proteins of the encoded by the shortest RNA segment 8. It is expressed early in of the influenza A virus. We observed that NS1 proteins of the encoded by the shortest RNA segment 8. It is expressed early in 1918 pandemic virus (A/Brevig Mission/1/18) and many avian viral replication cycle, and it is not a component of the virus 1918 pandemic virus (A/Brevig Mission/1/18) and many avian viral replication cycle, and it is not a component of the virus influenza A viruses contain a consensus Src homology 3 (SH3) particle (2). Instead, NS1 is a multifunctional virulence factor influenza A viruses contain a consensus Src homology 3 (SH3) particle (2). Instead, NS1 is a multifunctional virulence factor domain-binding motif. Screening of a comprehensive human that promotes virus replication in the host cell and helps to domain-binding motif. Screening of a comprehensive human that promotes virus replication in the host cell and helps to SH3 phage library revealed the N-terminal SH3 of Crk and CrkL evade antiviral immunity (3–5). In particular, NS1 uses several SH3 phage library revealed the N-terminal SH3 of Crk and CrkL evade antiviral immunity (3–5). In particular, NS1 uses several as the preferred binding partners. Studies with recombinant mechanisms to prevent suppression of influenza A virus repli- as the preferred binding partners. Studies with recombinant mechanisms to prevent suppression of influenza A virus repli- proteins confirmed avid binding of NS1 proteins of the 1918 cation by the type I interferon system of the host. proteins confirmed avid binding of NS1 proteins of the 1918 cation by the type I interferon system of the host. virus and a representative avian H7N3 strain to Crk/CrkL SH3 Recent studies have demonstrated that during influenza A virus and a representative avian H7N3 strain to Crk/CrkL SH3 Recent studies have demonstrated that during influenza A but not to other SH3 domains tested, including p85␣ and p85␤. virus infection NS1 protein activates the phosphatidylinositol but not to other SH3 domains tested, including p85␣ and p85␤. virus infection NS1 protein activates the phosphatidylinositol Endogenous CrkL readily co-precipitated NS1 from cells 3-kinase (PI3K)3 signaling pathway, apparently via its associa- Endogenous CrkL readily co-precipitated NS1 from cells 3-kinase (PI3K)3 signaling pathway, apparently via its associa- infected with the H7N3 virus. In transfected cells association tion with the p85 regulatory subunit of PI3K (6–9). Activation infected with the H7N3 virus. In transfected cells association tion with the p85 regulatory subunit of PI3K (6–9). Activation with CrkL was observed for NS1 of the 1918 and H7N3 viruses of the PI3K pathway seems to be important for influenza A virus with CrkL was observed for NS1 of the 1918 and H7N3 viruses of the PI3K pathway seems to be important for influenza A virus but not A/Udorn/72 or A/WSN/33 NS1 lacking this sequence replication, because in cell culture studies recombinant viruses but not A/Udorn/72 or A/WSN/33 NS1 lacking this sequence replication, because in cell culture studies recombinant viruses motif. SH3 binding was dispensable for suppression of inter- with mutations that prevented binding of NS1 to p85 formed motif. SH3 binding was dispensable for suppression of inter- with mutations that prevented binding of NS1 to p85 formed feron-induced gene expression by NS1 but was associated much smaller plaques and grew to 10-fold lower titers than the feron-induced gene expression by NS1 but was associated much smaller plaques and grew to 10-fold lower titers than the with enhanced phosphatidylinositol 3-kinase signaling, as wild-type virus (9). Moreover, compounds that inhibit PI3K can with enhanced phosphatidylinositol 3-kinase signaling, as wild-type virus (9). Moreover, compounds that inhibit PI3K can evidenced by increased Akt phosphorylation. Thus, the Span- strongly suppress influenza A virus replication (7, 9, 10). evidenced by increased Akt phosphorylation. Thus, the Span- strongly suppress influenza A virus replication (7, 9, 10). ish Flu virus resembles avian influenza A viruses in its ability Hale et al. (9) showed that the tyrosine residue 89 (Tyr-89) of ish Flu virus resembles avian influenza A viruses in its ability Hale et al. (9) showed that the tyrosine residue 89 (Tyr-89) of to recruit Crk/CrkL to modulate host cell signaling. NS1 protein serves a critical role in mediating binding to p85␤. to recruit Crk/CrkL to modulate host cell signaling. NS1 protein serves a critical role in mediating binding to p85␤. This tyrosine lies in the context similar to a YXNM motif, which This tyrosine lies in the context similar to a YXNM motif, which upon tyrosine phosphorylation can serve as a high affinity bind- upon tyrosine phosphorylation can serve as a high affinity bind- Pandemic as well as seasonal outbreaks of influenza A virus ing site for the SH2 domain of p85 (11), but apparently NS1 Pandemic as well as seasonal outbreaks of influenza A virus ing site for the SH2 domain of p85 (11), but apparently NS1 represent major threats to global public health. In the last cen- interacts with p85␤ in an SH2-independent manner (12). In represent major threats to global public health. In the last cen- interacts with p85␤ in an SH2-independent manner (12). In tury three major pandemics have occurred, in 1918, 1957, and addition, p85 contains an SH3 domain, and Zhou and co-work- tury three major pandemics have occurred, in 1918, 1957, and addition, p85 contains an SH3 domain, and Zhou and co-work- 1968, caused by H1N1 (Spanish flu), H2N2 (Asian flu), and ers (6, 13) have suggested that a PXXP sequence in NS1, which 1968, caused by H1N1 (Spanish flu), H2N2 (Asian flu), and ers (6, 13) have suggested that a PXXP sequence in NS1, which H3N2 (Hong Kong flu) viruses, respectively. Of these, the Span- resembles the consensus of an SH3-binding motif (see below), H3N2 (Hong Kong flu) viruses, respectively. Of these, the Span- resembles the consensus of an SH3-binding motif (see below), ish flu was the most severe and is estimated to have caused over may also contribute to the p85 interaction. ish flu was the most severe and is estimated to have caused over may also contribute to the p85 interaction. 40 million deaths worldwide (1). Recent human infections by SH3 domains are small protein modules that mediate inter- 40 million deaths worldwide (1). Recent human infections by SH3 domains are small protein modules that mediate inter- highly pathogenic H5N1 avian influenza A viruses have and intramolecular protein interactions and are often found in highly pathogenic H5N1 avian influenza A viruses have and intramolecular protein interactions and are often found in increased the concern that another global pandemic may occur. proteins regulating cellular signaling pathways, cytoskeletal increased the concern that another global pandemic may occur. proteins regulating cellular signaling pathways, cytoskeletal Influenza A virus belongs to the Orthomyxoviridae family of organization, and membrane trafficking (14, 15). SH3 domains Influenza A virus belongs to the Orthomyxoviridae family of organization, and membrane trafficking (14, 15). SH3 domains enveloped viruses. Its genome is organized into eight single- recognize short proline-rich sequences, which are typically enveloped viruses. Its genome is organized into eight single- recognize short proline-rich sequences, which are typically stranded, negative-sense RNA segments that code for 11 iden- characterized by (ϩ)-X⌽PXXP (class I) or PX⌽PX-(ϩ) (class II) stranded, negative-sense RNA segments that code for 11 iden- characterized by (ϩ)-X⌽PXXP (class I) or PX⌽PX-(ϩ) (class II) consensus sequences (where X is any amino acid; (ϩ) indicates consensus sequences (where X is any amino acid; (ϩ) indicates a positively charged residue; and ⌽ indicates a hydrophobic a positively charged residue; and ⌽ indicates a hydrophobic * This work was supported in part by grants (to K. S. and I. J.) from the Acad- * This work was supported in part by grants (to K. S. and I. J.) from the Acad- emy of Finland and the Sigrid Juselius Foundation and a grant from the residue) (15–17). Since the discovery that the human immuno- emy of Finland and the Sigrid Juselius Foundation and a grant from the residue) (15–17). Since the discovery that the human immuno- Helsinki University Hospital (to K. S.). The costs of publication of this article deficiency virus type 1 pathogenicity factor Nef regulates the Helsinki University Hospital (to K. S.). The costs of publication of this article deficiency virus type 1 pathogenicity factor Nef regulates the were defrayed in part by the payment of page charges. This article must were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 The abbreviations used are: SH3, Src homology 3; PI3K, phosphatidylinositol Section 1734 solely to indicate this fact. 3 The abbreviations used are: SH3, Src homology 3; PI3K, phosphatidylinositol 1 Supported by the Helsinki Biomedical Graduate School. 3-kinase; IFN, interferon; MBP, maltose-binding protein; TBS, Tris-buffered 1 Supported by the Helsinki Biomedical Graduate School. 3-kinase; IFN, interferon; MBP, maltose-binding protein; TBS, Tris-buffered 2 To whom correspondence should be addressed. Tel.: 358-9-191-26770; Fax: saline; GST, glutathione S-transferase; HA, hemagglutinin; ISRE, interferon- 2 To whom correspondence should be addressed. Tel.: 358-9-191-26770; Fax: saline; GST, glutathione S-transferase; HA, hemagglutinin; ISRE, interferon- 358-9-191-26491; E-mail: [email protected]. stimulated response element; WT, wild type. 358-9-191-26491; E-mail: [email protected]. stimulated response element; WT, wild type.

FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5719 FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5719 Enhanced PI3K Activation by NS1 via SH3 Binding Enhanced PI3K Activation by NS1 via SH3 Binding host cell via binding to SH3 domains of Src family protein cDNAs into pGEX-4T1 (GE Healthcare) and pMAL-c2x (New host cell via binding to SH3 domains of Src family protein cDNAs into pGEX-4T1 (GE Healthcare) and pMAL-c2x (New kinases (18), SH3 domain binding capacity has been demon- England Biolabs) vectors, respectively. To generate the plasmid kinases (18), SH3 domain binding capacity has been demon- England Biolabs) vectors, respectively. To generate the plasmid strated for many other proteins encoded by viral as well as bac- p85␣-BP, an oligonucleotide duplex encoding for the peptide strated for many other proteins encoded by viral as well as bac- p85␣-BP, an oligonucleotide duplex encoding for the peptide terial pathogens (19–24). CLNCFRPLPPLPPPPR (30) was inserted into the polylinker of terial pathogens (19–24). CLNCFRPLPPLPPPPR (30) was inserted into the polylinker of We noted that, unlike most other NS1 proteins from human pMAL-c2x. We noted that, unlike most other NS1 proteins from human pMAL-c2x. strains of influenza, the NS1 sequence of the 1918 pandemic A DNA fragment encoding for a 123-amino acid biotin strains of influenza, the NS1 sequence of the 1918 pandemic A DNA fragment encoding for a 123-amino acid biotin influenza virus (A/Brevig Mission/1/18/H1N1 (25)) contains a acceptor domain from Propionibacterium shermanii transcar- influenza virus (A/Brevig Mission/1/18/H1N1 (25)) contains a acceptor domain from Propionibacterium shermanii transcar- perfect class II consensus SH3-binding sequence, and in this boxylase (start, MKLK; end, IKIG) was PCR-amplified from the perfect class II consensus SH3-binding sequence, and in this boxylase (start, MKLK; end, IKIG) was PCR-amplified from the regard it resembles many avian strains of influenza (26). PinPoint-Xa1 T-vector (Promega) and inserted between the regard it resembles many avian strains of influenza (26). PinPoint-Xa1 T-vector (Promega) and inserted between the We have recently generated an essentially complete (n ϭ GST gene and the multiple cloning site of pGEX-4T1 to gener- We have recently generated an essentially complete (n ϭ GST gene and the multiple cloning site of pGEX-4T1 to gener- 296) collection of human SH3 domains in the form of a phage ate pGEX-PP. Codon optimized cDNAs for the SH3 domains of 296) collection of human SH3 domains in the form of a phage ate pGEX-PP. Codon optimized cDNAs for the SH3 domains of display library to allow comprehensive and unbiased identifica- Crk, CrkL, p85␣, p85␤, and Eps8L1 derived from the human display library to allow comprehensive and unbiased identifica- Crk, CrkL, p85␣, p85␤, and Eps8L1 derived from the human tion of preferred SH3 partners for cellular and viral ligand pro- SH3 library (27) were subsequently cloned in-frame after the tion of preferred SH3 partners for cellular and viral ligand pro- SH3 library (27) were subsequently cloned in-frame after the teins of interest (27). In this study we have made use of this biotin acceptor domain in pGEX-PP. To generate C-terminally teins of interest (27). In this study we have made use of this biotin acceptor domain in pGEX-PP. To generate C-terminally novel research tool to identify the Crk family adapter proteins biotinylated Crk and CrkL expression constructs, human CrkII novel research tool to identify the Crk family adapter proteins biotinylated Crk and CrkL expression constructs, human CrkII as high affinity ligands for the NS1 protein of the 1918 virus. and CrkL cDNAs with stop codons replaced by a KpnI site were as high affinity ligands for the NS1 protein of the 1918 virus. and CrkL cDNAs with stop codons replaced by a KpnI site were EXPERIMENTAL PROCEDURES fused with the above-described biotin acceptor domain frag- EXPERIMENTAL PROCEDURES fused with the above-described biotin acceptor domain frag- ment in pEBB. ment in pEBB. Cells and Viral Infections—Human embryonic kidney ISRE-Luc reporter plasmid contains a 30-bp interferon- Cells and Viral Infections—Human embryonic kidney ISRE-Luc reporter plasmid contains a 30-bp interferon- 293FT, human hepatocellular carcinoma Huh-7, and human stimulated response element-containing fragment from the 293FT, human hepatocellular carcinoma Huh-7, and human stimulated response element-containing fragment from the A549 lung carcinoma cell lines were maintained in Dulbecco’s ISG15 gene (31) in front of a minimal thymidine kinase pro- A549 lung carcinoma cell lines were maintained in Dulbecco’s ISG15 gene (31) in front of a minimal thymidine kinase pro- modified Eagle’s medium high glucose supplemented with 0.6 modified Eagle’s medium high glucose supplemented with 0.6 ␮ ␮ moter driving firefly luciferase expression (obtained from J. ␮ ␮ moter driving firefly luciferase expression (obtained from J. g/ml penicillin, 60 g/ml streptomycin, 10% fetal bovine Darnell Jr., Rockefeller University, New York). As a control g/ml penicillin, 60 g/ml streptomycin, 10% fetal bovine Darnell Jr., Rockefeller University, New York). As a control serum, and 2 mM glutamine. Influenza A virus strains A/mal- serum, and 2 mM glutamine. Influenza A virus strains A/mal- for transfection efficiency and cell viability, we used the plas- for transfection efficiency and cell viability, we used the plas- lard/Netherlands/12/2000 (H7N3) and A/Udorn/72 (H3N2) lard/Netherlands/12/2000 (H7N3) and A/Udorn/72 (H3N2) mid pcDNA-Renilla, which was created by inserting Renilla mid pcDNA-Renilla, which was created by inserting Renilla were grown in 11-day-old embryonated eggs, and the virus were grown in 11-day-old embryonated eggs, and the virus luciferase cDNA from pRL-null (Promega) into pcDNA3.1/ luciferase cDNA from pRL-null (Promega) into pcDNA3.1/ stock was aliquoted and stored at Ϫ70 °C. The hemagglutina- stock was aliquoted and stored at Ϫ70 °C. The hemagglutina- Hygro vector (Invitrogen). Hygro vector (Invitrogen). tion titers of the viruses were 256, and their infectivity in A549 tion titers of the viruses were 256, and their infectivity in A549 Antibodies and Other Reagents—The following antibodies Antibodies and Other Reagents—The following antibodies cells was 1 ϫ 107 and 2 ϫ 107 plaque-forming units per ml, cells was 1 ϫ 107 and 2 ϫ 107 plaque-forming units per ml, were used in this study: mouse anti-Myc (Sigma), mouse anti- were used in this study: mouse anti-Myc (Sigma), mouse anti- respectively. Virus infection of A549 cells was carried out in respectively. Virus infection of A549 cells was carried out in CrkL (Upstate), mouse anti-phospho-Akt(Ser-473) (Cell CrkL (Upstate), mouse anti-phospho-Akt(Ser-473) (Cell Dulbecco’s modified Eagle’s medium supplemented with 2% Dulbecco’s modified Eagle’s medium supplemented with 2% Signaling Technology), rabbit anti-NP (32), mouse anti-he- Signaling Technology), rabbit anti-NP (32), mouse anti-he- fetal bovine serum and antibiotics for 20 h at a multiplicity of fetal bovine serum and antibiotics for 20 h at a multiplicity of magglutinin (HA, Santa Cruz Biotechnology), and guinea pig magglutinin (HA, Santa Cruz Biotechnology), and guinea pig infection of 5 plaque-forming units/cell. infection of 5 plaque-forming units/cell. 35 anti-NS1 (28). Streptavidin IRDye800CW, IRDye 800CW 35 anti-NS1 (28). Streptavidin IRDye800CW, IRDye 800CW To metabolically label virus-infected cells with [ S]methi- To metabolically label virus-infected cells with [ S]methi- onine, the cells were washed with and changed into methio- goat anti-mouse IgG, and IRDye680 goat anti-mouse IgG onine, the cells were washed with and changed into methio- goat anti-mouse IgG, and IRDye680 goat anti-mouse IgG nine-free media supplemented with 2% fetal bovine serum and were from LI-COR Biotechnology. Secondary horseradish nine-free media supplemented with 2% fetal bovine serum and were from LI-COR Biotechnology. Secondary horseradish 0.5 mCi of [35S]Met (GE Healthcare) 4 h after infection and peroxidase-conjugated anti-guinea pig antibodies were from 0.5 mCi of [35S]Met (GE Healthcare) 4 h after infection and peroxidase-conjugated anti-guinea pig antibodies were from grown for an additional 16 h. At 20 h after infection cells were Jackson ImmunoResearch. grown for an additional 16 h. At 20 h after infection cells were Jackson ImmunoResearch. collected, washed twice with cold phosphate-buffered saline, Recombinant Proteins and Binding Assays—GST and MBP collected, washed twice with cold phosphate-buffered saline, Recombinant Proteins and Binding Assays—GST and MBP and lysed. fusion proteins expressed from pGEX-4T1 and pMAL-c2x pro- and lysed. fusion proteins expressed from pGEX-4T1 and pMAL-c2x pro- Plasmid Constructs—A synthetic gene fragment encoding tein expression vectors were purified using glutathione-Sepha- Plasmid Constructs—A synthetic gene fragment encoding tein expression vectors were purified using glutathione-Sepha- A/Brevig Mission/1/18 NS1 was purchased from GENEART rose 4B (GE Healthcare) or amylose resin (New England Bio- A/Brevig Mission/1/18 NS1 was purchased from GENEART rose 4B (GE Healthcare) or amylose resin (New England Bio- (Regensburg, Germany). A/mallard/Netherlands/12/2000/ labs), according to the manufacturer’s instructions. Screening (Regensburg, Germany). A/mallard/Netherlands/12/2000/ labs), according to the manufacturer’s instructions. Screening H7N3 cDNA was cloned from total cellular RNA of virus-in- of the human SH3 phage library using the GST-NS1 fusion H7N3 cDNA was cloned from total cellular RNA of virus-in- of the human SH3 phage library using the GST-NS1 fusion fected cells by standard methods. A/Udorn/72 and A/WSN/33 proteins was done as described previously (27). Recombinant fected cells by standard methods. A/Udorn/72 and A/WSN/33 proteins was done as described previously (27). Recombinant NS1 inserts were derived from pcDNA3.1-based constructs protein binding assay was done as in Ka¨rkka¨inen et al. (27) with NS1 inserts were derived from pcDNA3.1-based constructs protein binding assay was done as in Ka¨rkka¨inen et al. (27) with already described (28). To generate the mammalian NS1 some modifications. MBP-NS1 proteins, MBP-p85␣-BP, or already described (28). To generate the mammalian NS1 some modifications. MBP-NS1 proteins, MBP-p85␣-BP, or expression vectors, these fragments were inserted into the plain MBP was coated on 96-well plates (200 ng/well). Wells expression vectors, these fragments were inserted into the plain MBP was coated on 96-well plates (200 ng/well). Wells EF1␣ enhancer-driven vector pEBB-mycN by PCR-mediated were blocked with 1.5% bovine serum albumin in Tris-buffered EF1␣ enhancer-driven vector pEBB-mycN by PCR-mediated were blocked with 1.5% bovine serum albumin in Tris-buffered cloning and confirmed by DNA sequencing. pEBB-mycN is a saline (TBS) for 1 h and washed twice with TBS ϩ 0.05% Tween cloning and confirmed by DNA sequencing. pEBB-mycN is a saline (TBS) for 1 h and washed twice with TBS ϩ 0.05% Tween derivative of pEBB (from Bruce Mayer, University of Connect- 20 (TBST). MBP proteins were then incubated with 2-fold dilu- derivative of pEBB (from Bruce Mayer, University of Connect- 20 (TBST). MBP proteins were then incubated with 2-fold dilu- icut) (29), in which translation starts upstream of the insert to tions of GST-biotin-SH3 domains in TBS for 1.5 h. Wells were icut) (29), in which translation starts upstream of the insert to tions of GST-biotin-SH3 domains in TBS for 1.5 h. Wells were include a Myc epitope-containing peptide (MEQKLISEED- then washed three times with TBST, followed by a 1-h incuba- include a Myc epitope-containing peptide (MEQKLISEED- then washed three times with TBST, followed by a 1-h incuba- LGS) at the N terminus. Codon changes to A/Brevig and tion with streptavidin-biotinylated horseradish peroxidase LGS) at the N terminus. Codon changes to A/Brevig and tion with streptavidin-biotinylated horseradish peroxidase A/Mallard NS1 gene were generated by overlap PCR mutagen- complex (1:2000 dilution in TBS; GE Healthcare). After three A/Mallard NS1 gene were generated by overlap PCR mutagen- complex (1:2000 dilution in TBS; GE Healthcare). After three esis, cloned into pEBB-mycN, and verified by sequencing. Bac- washes with TBST, 50 ␮l of substrate reagent ABTS Single esis, cloned into pEBB-mycN, and verified by sequencing. Bac- washes with TBST, 50 ␮l of substrate reagent ABTS Single terial GST and MBP fusion protein expression vectors for NS1 Solution (Invitrogen) was added, and the absorbance at 405 nm terial GST and MBP fusion protein expression vectors for NS1 Solution (Invitrogen) was added, and the absorbance at 405 nm proteins were constructed by inserting the corresponding NS1 was measured 20 min later. proteins were constructed by inserting the corresponding NS1 was measured 20 min later.

5720 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 9•FEBRUARY 29, 2008 5720 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 9•FEBRUARY 29, 2008 Enhanced PI3K Activation by NS1 via SH3 Binding Enhanced PI3K Activation by NS1 via SH3 Binding

Protein Pulldowns and Western Blots—For protein pulldown Protein Pulldowns and Western Blots—For protein pulldown experiments, 293FT cells were transfected by standard calcium experiments, 293FT cells were transfected by standard calcium phosphate precipitation method with 10 ␮g of NS1 expression phosphate precipitation method with 10 ␮g of NS1 expression vectors in 10-cm plates. For avidin pulldown experiments cells vectors in 10-cm plates. For avidin pulldown experiments cells were transfected also with 3 ␮g of Crk or CrkL expression con- were transfected also with 3 ␮g of Crk or CrkL expression con- structs encoding for C-terminally biotinylated proteins. 48 h structs encoding for C-terminally biotinylated proteins. 48 h after transfection, cells were lysed on ice with 1% Nonidet P-40 after transfection, cells were lysed on ice with 1% Nonidet P-40 lysis buffer (150 mM NaCl; 50 mM Tris-HCl, pH 7.9; 1% Nonidet lysis buffer (150 mM NaCl; 50 mM Tris-HCl, pH 7.9; 1% Nonidet P-40). Cell lysates were used for immunoprecipitation with P-40). Cell lysates were used for immunoprecipitation with anti-CrkL antibody and Dynabeads protein A magnetic beads anti-CrkL antibody and Dynabeads protein A magnetic beads (Invitrogen) or avidin pulldowns with Tetralink tetrameric avi- (Invitrogen) or avidin pulldowns with Tetralink tetrameric avi- din resin (Promega). To examine the phosphorylation status of din resin (Promega). To examine the phosphorylation status of Akt, Huh-7 cells in 6-well plates were transfected with 4 ␮gof Akt, Huh-7 cells in 6-well plates were transfected with 4 ␮gof Myc-tagged NS1 expression plasmids or empty plasmid using Myc-tagged NS1 expression plasmids or empty plasmid using Lipofectamine 2000 (Invitrogen) according to the manufactur- Lipofectamine 2000 (Invitrogen) according to the manufactur- er’s instructions. 48 h after transfection cells were lysed on ice er’s instructions. 48 h after transfection cells were lysed on ice with 1% Nonidet P-40 lysis buffer, and lysates were used for with 1% Nonidet P-40 lysis buffer, and lysates were used for Western blotting to detect phospho-Akt or myc-NS1. Immo- Western blotting to detect phospho-Akt or myc-NS1. Immo- bilon Western chemiluminescent horseradish peroxidase sub- bilon Western chemiluminescent horseradish peroxidase sub- strate (Millipore) or Odyssey infrared imaging system (LI-COR strate (Millipore) or Odyssey infrared imaging system (LI-COR Biosciences) was used for detection. Biosciences) was used for detection. Reporter Gene Assays—To measure the activation of the ISRE Reporter Gene Assays—To measure the activation of the ISRE promoter, Huh-7 cells were transfected in 12-well plates with promoter, Huh-7 cells were transfected in 12-well plates with 0.2 ␮g of ISRE-Luc, 5 ng of pcDNA-Renilla, and 1 ␮gofNS1 0.2 ␮g of ISRE-Luc, 5 ng of pcDNA-Renilla, and 1 ␮gofNS1 expression vectors or empty vector using Lipofectamine 2000 expression vectors or empty vector using Lipofectamine 2000 (Invitrogen). 22 h post-transfection, the reporter gene was (Invitrogen). 22 h post-transfection, the reporter gene was ␤ FIGURE 1. Amino acid sequences of selected influenza A virus NS1 pro- ␤ FIGURE 1. Amino acid sequences of selected influenza A virus NS1 pro- induced by treatment with 100 IU/ml of IFN- (Betaferon, teins. A, alignment of the complete influenza A virus NS1 sequences of A/Bre- induced by treatment with 100 IU/ml of IFN- (Betaferon, teins. A, alignment of the complete influenza A virus NS1 sequences of A/Bre- Schering) for 7 h. Cells were lysed with Passive Lysis Buffer vig Mission/1/18/H1N1 (Brevig) and A/Udorn/72/H3N2 (Udorn) sequences. Schering) for 7 h. Cells were lysed with Passive Lysis Buffer vig Mission/1/18/H1N1 (Brevig) and A/Udorn/72/H3N2 (Udorn) sequences. (Promega), and the luciferase activity was measured using dual- Residues in Udorn that differ from the corresponding amino acids in Brevig (Promega), and the luciferase activity was measured using dual- Residues in Udorn that differ from the corresponding amino acids in Brevig are highlighted in gray. Tyrosine (position 89) and proline (positions 164, 167, are highlighted in gray. Tyrosine (position 89) and proline (positions 164, 167, luciferase assay system (Promega) and Sirius luminometer 213, and 216) previously implicated in binding to the SH2 and SH3 domains of luciferase assay system (Promega) and Sirius luminometer 213, and 216) previously implicated in binding to the SH2 and SH3 domains of (Berthold detection systems). Renilla luciferase construct was PI3K-p85 (6, 9) are underlined. The region containing a consensus SH3-bind- (Berthold detection systems). Renilla luciferase construct was PI3K-p85 (6, 9) are underlined. The region containing a consensus SH3-bind- ing motif in the Brevig sequence is boxed. B, shown are amino acid sequences ing motif in the Brevig sequence is boxed. B, shown are amino acid sequences used as an internal control to normalize relative luciferase within in the region boxed in A in NS1 proteins from the indicated influenza A used as an internal control to normalize relative luciferase within in the region boxed in A in NS1 proteins from the indicated influenza A activity. viruses, as well as chicken (based on 1996 isolates) and human (based on 2005 activity. viruses, as well as chicken (based on 1996 isolates) and human (based on 2005 isolates) consensus sequences. The sequence Px⌽Pxϩ (where x denotes any isolates) consensus sequences. The sequence Px⌽Pxϩ (where x denotes any RESULTS amino acid, ⌽ indicates a hydrophobic residue, and ϩ indicates an arginine or RESULTS amino acid, ⌽ indicates a hydrophobic residue, and ϩ indicates an arginine or a lysine) indicates a class II SH3-binding consensus motif (critical amino acids a lysine) indicates a class II SH3-binding consensus motif (critical amino acids Consensus SH3-binding Motif in NS1—To look for potential in boldface). Consensus SH3-binding Motif in NS1—To look for potential in boldface). virally encoded ligands for cellular SH3 proteins, we used the virally encoded ligands for cellular SH3 proteins, we used the ScanProsite search engine to identify consensus SH3 domain- encoded by A/Brevig and an avian strain containing the same ScanProsite search engine to identify consensus SH3 domain- encoded by A/Brevig and an avian strain containing the same binding motifs in viral protein sequences in the Swiss-Prot/ consensus motif (A/mallard/Netherlands/12/00/H7N3; binding motifs in viral protein sequences in the Swiss-Prot/ consensus motif (A/mallard/Netherlands/12/00/H7N3; TrEMBL data base. One interesting protein that was noted to A/Mallard below) were expressed as GST fusion proteins in TrEMBL data base. One interesting protein that was noted to A/Mallard below) were expressed as GST fusion proteins in contain a perfect class II SH3-binding motif was NS1 of the Escherichia coli and used as ligands for affinity screening of our contain a perfect class II SH3-binding motif was NS1 of the Escherichia coli and used as ligands for affinity screening of our 1918 pandemic influenza A virus (A/Brevig Mission/1/18/ comprehensive human SH3 phage display library (27). Both 1918 pandemic influenza A virus (A/Brevig Mission/1/18/ comprehensive human SH3 phage display library (27). Both H1N1; A/Brevig below). Analysis of NS1 protein sequences proteins bound avidly to SH3 clones in the library as compared H1N1; A/Brevig below). Analysis of NS1 protein sequences proteins bound avidly to SH3 clones in the library as compared from other strains of influenza A revealed that this sequence with plain GST protein used as a control for nonspecific bind- from other strains of influenza A revealed that this sequence with plain GST protein used as a control for nonspecific bind- motif is very common among avian influenza A viruses but only ing (not shown). Sequencing of the phagemid genomes motif is very common among avian influenza A viruses but only ing (not shown). Sequencing of the phagemid genomes rarely found in viruses isolated from humans (Fig. 1). In addi- obtained after a single round of affinity selection with these NS1 rarely found in viruses isolated from humans (Fig. 1). In addi- obtained after a single round of affinity selection with these NS1 tion to A/Brevig, only three other human-derived NS1 proteins revealed that more than 90% of the phages contained tion to A/Brevig, only three other human-derived NS1 proteins revealed that more than 90% of the phages contained sequences containing this motif could be found from the NCBI the N-terminal SH3 domain of the adapter protein Crk or its sequences containing this motif could be found from the NCBI the N-terminal SH3 domain of the adapter protein Crk or its Influenza Virus Resource data base. Notably, two of these close homologue CrkL. Influenza Virus Resource data base. Notably, two of these close homologue CrkL. viruses represented recent zoonotic transmissions from birds To confirm and study in more detail the NS1/SH3 interac- viruses represented recent zoonotic transmissions from birds To confirm and study in more detail the NS1/SH3 interac- with an H5N1 virus (A/Hong Kong/481/97/H5N1 (33)) and an tions revealed by phage screening, we generated recombinant with an H5N1 virus (A/Hong Kong/481/97/H5N1 (33)) and an tions revealed by phage screening, we generated recombinant H7N3 virus (A/Canada/rv504/2004/H7N3 (26)). Accordingly, GST fusion proteins of the N-terminal SH3 domains of Crk and H7N3 virus (A/Canada/rv504/2004/H7N3 (26)). Accordingly, GST fusion proteins of the N-terminal SH3 domains of Crk and this sequence motif is not present in the NS1 proteins of human CrkL. Because the SH3 of p85 has been suggested to bind to this sequence motif is not present in the NS1 proteins of human CrkL. Because the SH3 of p85 has been suggested to bind to influenza A viruses commonly used for laboratory studies, such PXXP sequences found in many influenza A NS1 proteins (6) influenza A viruses commonly used for laboratory studies, such PXXP sequences found in many influenza A NS1 proteins (6) as A/WSN/33 (H1N1), A/PR/8/34 (H1N1), and A/Udorn/72 (see Fig. 1) we also generated GST-SH3 fusion proteins of p85␣ as A/WSN/33 (H1N1), A/PR/8/34 (H1N1), and A/Udorn/72 (see Fig. 1) we also generated GST-SH3 fusion proteins of p85␣ (H3N2) (see Fig. 1). and p85␤. In addition, the SH3 of Eps8L1, which is known to (H3N2) (see Fig. 1). and p85␤. In addition, the SH3 of Eps8L1, which is known to Identification of SH3 Partners of NS1—To examine their prefer atypical PXXDY-containing ligands (34), was included as Identification of SH3 Partners of NS1—To examine their prefer atypical PXXDY-containing ligands (34), was included as SH3 binding potential and preferences, NS1 proteins a negative control. Between the GST and SH3 moieties in these SH3 binding potential and preferences, NS1 proteins a negative control. Between the GST and SH3 moieties in these

FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5721 FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5721 Enhanced PI3K Activation by NS1 via SH3 Binding Enhanced PI3K Activation by NS1 via SH3 Binding

FIGURE 3. Co-precipitation of A/Brevig, A/Mallard, and A/Udorn NS1 pro- FIGURE 3. Co-precipitation of A/Brevig, A/Mallard, and A/Udorn NS1 pro- teins with full-length Crk and CrkL proteins from transfected cells. Myc- teins with full-length Crk and CrkL proteins from transfected cells. Myc- tagged expression vectors for the indicated NS1 proteins were transfected tagged expression vectors for the indicated NS1 proteins were transfected into 293FT cells together with a biotin-acceptor domain-tagged vector for Crk into 293FT cells together with a biotin-acceptor domain-tagged vector for Crk (C) or CrkL (L). Material precipitated with avidin-coated beads from lysates of (C) or CrkL (L). Material precipitated with avidin-coated beads from lysates of the co-transfected cells were analyzed by Western blotting using an anti-Myc the co-transfected cells were analyzed by Western blotting using an anti-Myc antibody or a labeled avidin reagent, as indicated. Aliquots of the lysates were antibody or a labeled avidin reagent, as indicated. Aliquots of the lysates were collected before avidin pulldown and subjected directly into Western blot collected before avidin pulldown and subjected directly into Western blot analysis (two bottom panels). analysis (two bottom panels).

Eps8L1 SH3 or CrkL SH3 in binding to the control wells coated Eps8L1 SH3 or CrkL SH3 in binding to the control wells coated with plain MBP. Although the absolute binding to Crk/CrkL with plain MBP. Although the absolute binding to Crk/CrkL SH3 domains was slightly weaker, very similar results were SH3 domains was slightly weaker, very similar results were obtained by using A/Brevig NS1 as the immobilized ligand obtained by using A/Brevig NS1 as the immobilized ligand (data not shown). In agreement with the lack of a consensus (data not shown). In agreement with the lack of a consensus SH3-binding motif in A/Udorn NS1, none of the tested SH3 SH3-binding motif in A/Udorn NS1, none of the tested SH3 domains showed measurable binding to Udorn NS1 protein domains showed measurable binding to Udorn NS1 protein (Fig. 2B). To confirm the functionality of the p85 SH3 domain (Fig. 2B). To confirm the functionality of the p85 SH3 domain proteins used in this study, we fused MBP with a peptide proteins used in this study, we fused MBP with a peptide FIGURE 2. Semi-quantitative analysis of binding of recombinant SH3 FIGURE 2. Semi-quantitative analysis of binding of recombinant SH3 domains to NS1 proteins. A/Mallard (A) and A/Udorn (B) NS1 proteins were (CLNCFRPLPPLPPPPR) that has been optimized for binding to domains to NS1 proteins. A/Mallard (A) and A/Udorn (B) NS1 proteins were (CLNCFRPLPPLPPPPR) that has been optimized for binding to expressed as MBP in E. coli and used to coat 96-well plates. These wells were p85␣ using phage display (30). Positive binding to this peptide expressed as MBP in E. coli and used to coat 96-well plates. These wells were p85␣ using phage display (30). Positive binding to this peptide incubated with 2-fold dilutions (ranging from 2.5 to 0.16 ␮M) of the indicated incubated with 2-fold dilutions (ranging from 2.5 to 0.16 ␮M) of the indicated recombinant SH3 domains expressed as biotinylated GST fusion proteins, under identical assay conditions (Fig. 2C) indicated that the recombinant SH3 domains expressed as biotinylated GST fusion proteins, under identical assay conditions (Fig. 2C) indicated that the followed by detection of binding with an enzymatically labeled streptavidin. failure of p85␣ and p85␤ SH3 domains to bind to the NS1 pro- followed by detection of binding with an enzymatically labeled streptavidin. failure of p85␣ and p85␤ SH3 domains to bind to the NS1 pro- As a control for nonspecific binding, additional wells were coated with plain teins of A/Mallard and A/Udorn (Fig. 2, A and B) as well as As a control for nonspecific binding, additional wells were coated with plain teins of A/Mallard and A/Udorn (Fig. 2, A and B) as well as MBP and similarly probed with CrkL SH3 domains (CrkL/MBP). In addition, an MBP and similarly probed with CrkL SH3 domains (CrkL/MBP). In addition, an artificial protein (p85␣-BP) consisting of MBP fused to a high affinity p85␣-SH3 A/Brevig and A/WSN (data not shown) was indeed a valid artificial protein (p85␣-BP) consisting of MBP fused to a high affinity p85␣-SH3 A/Brevig and A/WSN (data not shown) was indeed a valid ligand peptide (CLNCFRPLPPLPPPPR) (30) was used as an immobilized target result and not because of a general lack of functionality of our ligand peptide (CLNCFRPLPPLPPPPR) (30) was used as an immobilized target result and not because of a general lack of functionality of our protein to test binding of p85␣-SH3 and p85␤-SH3. As in A and B, wells coated protein to test binding of p85␣-SH3 and p85␤-SH3. As in A and B, wells coated with plain MBP (p85␣-SH3/MBP and p85␤/MBP) were used as controls (C). p85 SH3 proteins. with plain MBP (p85␣-SH3/MBP and p85␤/MBP) were used as controls (C). p85 SH3 proteins. Binding of Crk and CrkL to NS1 in Transfected Cells—To Binding of Crk and CrkL to NS1 in Transfected Cells—To extend these findings to full-length proteins expressed in extend these findings to full-length proteins expressed in fusion proteins, we inserted a biotin acceptor domain from P. human cells and to compare NS1-binding by Crk and CrkL, we fusion proteins, we inserted a biotin acceptor domain from P. human cells and to compare NS1-binding by Crk and CrkL, we shermanii transcarboxylase, which during expression in E. coli generated expression vectors for these proteins tagged at their shermanii transcarboxylase, which during expression in E. coli generated expression vectors for these proteins tagged at their becomes efficiently biotinylated at a single lysine residue, and N termini with the transcarboxylase biotin acceptor domain. As becomes efficiently biotinylated at a single lysine residue, and N termini with the transcarboxylase biotin acceptor domain. As can be conveniently detected with avidin-based reagents (35). in our previous studies on divergent protein interactions (36), can be conveniently detected with avidin-based reagents (35). in our previous studies on divergent protein interactions (36), As targets for these SH3 domains, we expressed maltose-bind- this strategy allowed sensitive and equal detection of the tagged As targets for these SH3 domains, we expressed maltose-bind- this strategy allowed sensitive and equal detection of the tagged ing protein (MBP) fusion constructs A/Udorn, A/Mallard, and Crk and CrkL proteins when co-transfected into 293FT cells ing protein (MBP) fusion constructs A/Udorn, A/Mallard, and Crk and CrkL proteins when co-transfected into 293FT cells A/Brevig NS1 proteins. The latter were coated onto the bottom together with NS1 proteins from different influenza A viruses A/Brevig NS1 proteins. The latter were coated onto the bottom together with NS1 proteins from different influenza A viruses of 96-well plates and probed with serial dilutions of biotinylated (Fig. 3). To ensure equal detection of the different NS1 proteins of 96-well plates and probed with serial dilutions of biotinylated (Fig. 3). To ensure equal detection of the different NS1 proteins Crk, CrkL, p85␣, p85␤, or Eps8L1 SH3 domains in an enzyme- used in this experiment, these expression constructs were Crk, CrkL, p85␣, p85␤, or Eps8L1 SH3 domains in an enzyme- used in this experiment, these expression constructs were linked immunosorbent assay-like sandwich assay. tagged with a Myc peptide epitope. Similar expression of all linked immunosorbent assay-like sandwich assay. tagged with a Myc peptide epitope. Similar expression of all As shown in Fig. 2, SH3 domains of Crk and CrkL bound NS1 proteins in the lysates of transfected cells and equal recov- As shown in Fig. 2, SH3 domains of Crk and CrkL bound NS1 proteins in the lysates of transfected cells and equal recov- avidly to A/Mallard NS1-coated wells, showing significant ery of the biotinylated Crk and CrkL proteins using avidin- avidly to A/Mallard NS1-coated wells, showing significant ery of the biotinylated Crk and CrkL proteins using avidin- binding signals even when tested at sub-micromolar concen- coated beads were observed, whereas the amounts of co-pre- binding signals even when tested at sub-micromolar concen- coated beads were observed, whereas the amounts of co-pre- trations (see Fig. 2A). By contrast, p85␣ and p85␤ SH3 domains cipitated NS1 proteins differed dramatically (Fig. 3, top panel). trations (see Fig. 2A). By contrast, p85␣ and p85␤ SH3 domains cipitated NS1 proteins differed dramatically (Fig. 3, top panel). were equally negative in binding to A/Mallard NS1 as was Both Crk and CrkL associated strongly with A/Brevig and were equally negative in binding to A/Mallard NS1 as was Both Crk and CrkL associated strongly with A/Brevig and

5722 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 9•FEBRUARY 29, 2008 5722 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 9•FEBRUARY 29, 2008 Enhanced PI3K Activation by NS1 via SH3 Binding Enhanced PI3K Activation by NS1 via SH3 Binding

the NS1 signal derived from a fraction of the total lysate equal- the NS1 signal derived from a fraction of the total lysate equal- ing 10% of the fraction used for the ␣-CrkL immunoprecipita- ing 10% of the fraction used for the ␣-CrkL immunoprecipita- tion. By contrast, no such signal was seen in anti-CrkL immu- tion. By contrast, no such signal was seen in anti-CrkL immu- noprecipitates from uninfected cells or from control (␣-HA) noprecipitates from uninfected cells or from control (␣-HA) immunoprecipitates from the A/Mallard-infected cells, thus immunoprecipitates from the A/Mallard-infected cells, thus confirming the specificity of the observed CrkL/NS1 confirming the specificity of the observed CrkL/NS1 co-precipitation. co-precipitation. A similar A549 infection experiment was carried out in A similar A549 infection experiment was carried out in which A/Udorn was included for comparison. Because of the which A/Udorn was included for comparison. Because of the sequence divergence of the A/Mallard and A/Udorn NS1 pro- sequence divergence of the A/Mallard and A/Udorn NS1 pro- teins, quantitative comparison of the expression levels of these teins, quantitative comparison of the expression levels of these proteins in their native form was not possible using any of the proteins in their native form was not possible using any of the immunological reagents that we tested (data not shown). immunological reagents that we tested (data not shown). Therefore, we chose to infect A549 cells with matched titers of Therefore, we chose to infect A549 cells with matched titers of A/Mallard and A/Udorn viruses, and subjected the infected A/Mallard and A/Udorn viruses, and subjected the infected cells to metabolic labeling with [35S]methionine. Because cells to metabolic labeling with [35S]methionine. Because A/Mallard and A/Udorn NS1 proteins contain a comparable A/Mallard and A/Udorn NS1 proteins contain a comparable number of methionine residues, their presence in anti-CrkL number of methionine residues, their presence in anti-CrkL immunoprecipitates could be accurately compared by autora- immunoprecipitates could be accurately compared by autora- diography (Fig. 4B). diography (Fig. 4B). In good agreement with the Western blotting data (Fig. 4A) In good agreement with the Western blotting data (Fig. 4A) an NS1-sized protein was co-precipitated by the anti-CrkL an NS1-sized protein was co-precipitated by the anti-CrkL antibody from A/Mallard-infected cells but not from unin- antibody from A/Mallard-infected cells but not from unin- fected cells (Fig. 4B). A faint signal corresponding to a slightly fected cells (Fig. 4B). A faint signal corresponding to a slightly larger protein (as expected for A/Udorn NS1) could also be larger protein (as expected for A/Udorn NS1) could also be detected from A/Udorn-infected cells in overexposures of the detected from A/Udorn-infected cells in overexposures of the autoradiogram shown in Fig. 4B and other similar experiments autoradiogram shown in Fig. 4B and other similar experiments (data not shown). Thus, despite its lack of direct CrkL binding (data not shown). Thus, despite its lack of direct CrkL binding capacity (Figs. 2 and 3), in infected cells A/Udorn NS1 might yet capacity (Figs. 2 and 3), in infected cells A/Udorn NS1 might yet FIGURE 4. Co-precipitation of NS1 with endogenous CrkL from influ- be weakly associated with CrkL. Equal infection of the cells with FIGURE 4. Co-precipitation of NS1 with endogenous CrkL from influ- be weakly associated with CrkL. Equal infection of the cells with enza A virus-infected cells. A, lysates from control A549 cells (Ϫ) and enza A virus-infected cells. A, lysates from control A549 cells (Ϫ) and cells infected with A/mallard/Netherlands/12/00/H7N3 virus (M) were the A/Mallard and A/Udorn viruses was confirmed by Western cells infected with A/mallard/Netherlands/12/00/H7N3 virus (M) were the A/Mallard and A/Udorn viruses was confirmed by Western subjected to immunoprecipitation (IP) with anti-CrkL (␣-CrkL) or control blotting using an antiserum against nucleoprotein, which subjected to immunoprecipitation (IP) with anti-CrkL (␣-CrkL) or control blotting using an antiserum against nucleoprotein, which (␣-HA) monoclonal antibodies. These precipitates and aliquots of the cor- shows high amino acid sequence conservation between differ- (␣-HA) monoclonal antibodies. These precipitates and aliquots of the cor- shows high amino acid sequence conservation between differ- responding lysates were analyzed by Western blotting using a polyclonal responding lysates were analyzed by Western blotting using a polyclonal anti-NS1 antiserum. B, lysates of [35S]methionine-labeled control A549 ent influenza A strains (Fig. 4B, right panel). Together these anti-NS1 antiserum. B, lysates of [35S]methionine-labeled control A549 ent influenza A strains (Fig. 4B, right panel). Together these cells (Ϫ) or similarly labeled cells infected with A/mallard/Netherlands/12/ data confirmed binding of endogenous CrkL to NS1 in virus- cells (Ϫ) or similarly labeled cells infected with A/mallard/Netherlands/12/ data confirmed binding of endogenous CrkL to NS1 in virus- 00/H7N3 (M) or A/Udorn/72 (U) viruses were subjected to immunoprecipi- 00/H7N3 (M) or A/Udorn/72 (U) viruses were subjected to immunoprecipi- tation with an anti-CrkL antibody, followed by SDS-PAGE and autoradiog- infected cells, and underscore the important role of the SH3 tation with an anti-CrkL antibody, followed by SDS-PAGE and autoradiog- infected cells, and underscore the important role of the SH3 raphy (left panel). The labeled lysates were also subjected to Western binding capacity of NS1 alleles like A/Mallard in promoting this raphy (left panel). The labeled lysates were also subjected to Western binding capacity of NS1 alleles like A/Mallard in promoting this blotting with an anti-nucleoprotein (NP) antibody (right panel). interaction. blotting with an anti-nucleoprotein (NP) antibody (right panel). interaction. Characterization of the Crk/CrkL SH3-binding Site in NS1— Characterization of the Crk/CrkL SH3-binding Site in NS1— A/Mallard NS1 proteins, but they failed to co-precipitate any To confirm that the class II SH3-binding motif in A/Mallard A/Mallard NS1 proteins, but they failed to co-precipitate any To confirm that the class II SH3-binding motif in A/Mallard A/Udorn NS1. Binding to A/WSN NS1 was equally negative and A/Brevig NS1 protein was indeed critical for their capacity A/Udorn NS1. Binding to A/WSN NS1 was equally negative and A/Brevig NS1 protein was indeed critical for their capacity (data not shown). Thus, in good agreement with the recombi- to bind to the Crk family proteins, we generated a series of point (data not shown). Thus, in good agreement with the recombi- to bind to the Crk family proteins, we generated a series of point nant protein data involving isolated Crk/CrkL SH3 domains, in mutated variants of A/Brevig NS1 shown in Fig. 5A. Mutant 1 nant protein data involving isolated Crk/CrkL SH3 domains, in mutated variants of A/Brevig NS1 shown in Fig. 5A. Mutant 1 transfected cells full-length Crk/CrkL proteins bound well to (M1) contained alanine residues in place of both of the PXXP- transfected cells full-length Crk/CrkL proteins bound well to (M1) contained alanine residues in place of both of the PXXP- NS1 proteins containing a class II SH3-binding consensus site defining proline residues of the class II consensus motif NS1 proteins containing a class II SH3-binding consensus site defining proline residues of the class II consensus motif but not detectably to NS1 proteins lacking this motif. (P212A/P215A). Mutant 2 (M2) contained a similar double ala- but not detectably to NS1 proteins lacking this motif. (P212A/P215A). Mutant 2 (M2) contained a similar double ala- CrkL-NS1 Complex in Influenza A Virus-infected Cells—Be- nine substitution but affected the additional PXXP sequence CrkL-NS1 Complex in Influenza A Virus-infected Cells—Be- nine substitution but affected the additional PXXP sequence cause of the similar NS1 binding profiles of Crk and CrkL, lack that is embedded within but is not part of the consensus SH3- cause of the similar NS1 binding profiles of Crk and CrkL, lack that is embedded within but is not part of the consensus SH3- of known differences in their cellular functions, and the avail- binding motif in A/Brevig NS1 (P213A/P216A). These proline of known differences in their cellular functions, and the avail- binding motif in A/Brevig NS1 (P213A/P216A). These proline ability of a good antibody against Crk, NS1 binding by CrkL was residues are conserved in NS1 proteins of most human and ability of a good antibody against Crk, NS1 binding by CrkL was residues are conserved in NS1 proteins of most human and chosen as the subject of our subsequent studies. To examine avian influenza viruses, including A/Udorn, and were pointed chosen as the subject of our subsequent studies. To examine avian influenza viruses, including A/Udorn, and were pointed whether binding of endogenous cellular CrkL with NS1 protein out by Shin et al. (6) as a potential docking site for p85-SH3. whether binding of endogenous cellular CrkL with NS1 protein out by Shin et al. (6) as a potential docking site for p85-SH3. produced during influenza A virus infection could be demon- Mutants 3 and 4 involved changes in the positively charged produced during influenza A virus infection could be demon- Mutants 3 and 4 involved changes in the positively charged strated, we infected A549 cells with A/Mallard and examined residue of the consensus motif. In M3 this charge was reversed strated, we infected A549 cells with A/Mallard and examined residue of the consensus motif. In M3 this charge was reversed the presence of NS1 in anti-CrkL immunoprecipitates from by a lysine to glutamic acid substitution (K217E), whereas the the presence of NS1 in anti-CrkL immunoprecipitates from by a lysine to glutamic acid substitution (K217E), whereas the lysates of these cells. As shown in Fig. 4A, a readily detectable lysine to arginine substitution in M4 (K217R) maintained this lysates of these cells. As shown in Fig. 4A, a readily detectable lysine to arginine substitution in M4 (K217R) maintained this amount of NS1 co-precipitated from the A/Mallard-infected functionally important positive charge. In fact, arginine is the amount of NS1 co-precipitated from the A/Mallard-infected functionally important positive charge. In fact, arginine is the cells using the ␣-CrkL antibody, corresponding in intensity to positively charged residue in most class II SH3-binding sites, cells using the ␣-CrkL antibody, corresponding in intensity to positively charged residue in most class II SH3-binding sites,

FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5723 FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5723 Enhanced PI3K Activation by NS1 via SH3 Binding Enhanced PI3K Activation by NS1 via SH3 Binding

FIGURE 5. Mutational analysis of NS1 residues involved in binding to cel- FIGURE 5. Mutational analysis of NS1 residues involved in binding to cel- lular CrkL. A, class II SH3-binding motif-containing region of wild-type A/Bre- lular CrkL. A, class II SH3-binding motif-containing region of wild-type A/Bre- vig NS1 protein (WT) and its derivatives (M1–M5) carrying the indicated muta- vig NS1 protein (WT) and its derivatives (M1–M5) carrying the indicated muta- tions are shown. B, Myc-tagged expression vectors for WT or mutated variants tions are shown. B, Myc-tagged expression vectors for WT or mutated variants of A/Brevig NS1 protein (M1–M5) or an empty control vector were transfected of A/Brevig NS1 protein (M1–M5) or an empty control vector were transfected into 293FT cells. Lysates of these cells were subjected to anti-CrkL immuno- into 293FT cells. Lysates of these cells were subjected to anti-CrkL immuno- precipitation (IP), and these precipitates along with aliquots of the original FIGURE 6. Functional analysis of the role of Crk/CrkL SH3 domain binding precipitation (IP), and these precipitates along with aliquots of the original FIGURE 6. Functional analysis of the role of Crk/CrkL SH3 domain binding lysates were analyzed by Western blotting with an anti-Myc antibody. capacity in regulation of cellular signaling pathways by NS1. A, Huh-7 lysates were analyzed by Western blotting with an anti-Myc antibody. capacity in regulation of cellular signaling pathways by NS1. A, Huh-7 cells were transfected with an ISRE-dependent firefly luciferase reporter vec- cells were transfected with an ISRE-dependent firefly luciferase reporter vec- tor together with an expression vector for A/Brevig (B), A/Mallard (M), or tor together with an expression vector for A/Brevig (B), A/Mallard (M), or but Crk and CrkL SH3 domains have a characteristic property A/Udorn (U) NS1 or SH3 binding-deficient mutants (M1 in Fig. 5) of A/Brevig but Crk and CrkL SH3 domains have a characteristic property A/Udorn (U) NS1 or SH3 binding-deficient mutants (M1 in Fig. 5) of A/Brevig (Bm) or A/Mallard (Mm) NS1 proteins, or an empty control plasmid vector (p). In (Bm) or A/Mallard (Mm) NS1 proteins, or an empty control plasmid vector (p). In of preferring a lysine residue in this position (37). Mutant 5 addition, a vector stably expressing Renilla luciferase was included in all cases of preferring a lysine residue in this position (37). Mutant 5 addition, a vector stably expressing Renilla luciferase was included in all cases (M5) contained a proline to threonine substitution (P215T) to monitor transfection efficiency and cell viability. After 22 h, one culture of (M5) contained a proline to threonine substitution (P215T) to monitor transfection efficiency and cell viability. After 22 h, one culture of cells transfected with the control vector was left untreated, and the other cells transfected with the control vector was left untreated, and the other affecting the first proline residue of the consensus motif, thus plates were stimulated for 7 h with 100 IU/ml of IFN-␤, followed by determi- affecting the first proline residue of the consensus motif, thus plates were stimulated for 7 h with 100 IU/ml of IFN-␤, followed by determi- mimicking the NS1 protein sequence in this region of A/Udorn nation of firefly and Renilla luciferase activities of lysates prepared from these mimicking the NS1 protein sequence in this region of A/Udorn nation of firefly and Renilla luciferase activities of lysates prepared from these (and practically all human influenza A virus NS1 proteins; see cells. ISRE-dependent gene expression is shown as mean values of the ratios (and practically all human influenza A virus NS1 proteins; see cells. ISRE-dependent gene expression is shown as mean values of the ratios of firefly and Renilla luciferase activities. B, Huh-7 cells were transfected with of firefly and Renilla luciferase activities. B, Huh-7 cells were transfected with Fig. 1). the NS1 or control expression vectors used in A plus a vector for A/WSN/33 Fig. 1). the NS1 or control expression vectors used in A plus a vector for A/WSN/33 The capacity of Myc epitope-tagged NS1 proteins carrying NS1 (W). 48 h later the cells were collected and analyzed for their PI3K path- The capacity of Myc epitope-tagged NS1 proteins carrying NS1 (W). 48 h later the cells were collected and analyzed for their PI3K path- these mutations to bind to endogenous CrkL protein in trans- way activity by Western blot analysis of their lysates using an antibody spe- these mutations to bind to endogenous CrkL protein in trans- way activity by Western blot analysis of their lysates using an antibody spe- cific for the phosphorylated form of Akt. Uniform expression of the NS1 pro- cific for the phosphorylated form of Akt. Uniform expression of the NS1 pro- fected 293FT cells was studied by a co-immunoprecipitation teins was confirmed by probing the blots with an anti-Myc tag antibody. fected 293FT cells was studied by a co-immunoprecipitation teins was confirmed by probing the blots with an anti-Myc tag antibody. assay. As shown in Fig. 5B, NS1 was equally expressed in all assay. As shown in Fig. 5B, NS1 was equally expressed in all transfected cells, but M1 and M3 that carried class II consensus the p85 subunit of PI3K and can be followed by examining the transfected cells, but M1 and M3 that carried class II consensus the p85 subunit of PI3K and can be followed by examining the motif-disrupting mutations could not be co-precipitated with phosphorylation status of the protein kinase Akt/PKB (6, 8, 41). motif-disrupting mutations could not be co-precipitated with phosphorylation status of the protein kinase Akt/PKB (6, 8, 41). CrkL. The single P215T change (M5) resembling NS1 proteins When Huh-7 cells were transfected with an ISRE-containing CrkL. The single P215T change (M5) resembling NS1 proteins When Huh-7 cells were transfected with an ISRE-containing like that of A/Udorn also abolished binding to CrkL. Instead, reporter plasmid together with an empty control expression like that of A/Udorn also abolished binding to CrkL. Instead, reporter plasmid together with an empty control expression association of M2 with CrkL was indistinguishable of unmodi- vector (p), a robust increase in luciferase activity was observed association of M2 with CrkL was indistinguishable of unmodi- vector (p), a robust increase in luciferase activity was observed fied A/Brevig NS1 (WT). In agreement with the published upon stimulation of these cells with interferon (IFN-␤) (com- fied A/Brevig NS1 (WT). In agreement with the published upon stimulation of these cells with interferon (IFN-␤) (com- binding preferences of Crk/CrkL SH3 (37), binding of M4 to pare the two leftmost bars in Fig. 6A). When a vector expressing binding preferences of Crk/CrkL SH3 (37), binding of M4 to pare the two leftmost bars in Fig. 6A). When a vector expressing CrkL was diminished ϳ50% compared with WT (Fig. 4B and NS1 of A/Brevig (B), A/Mallard (M), or A/Udorn (U) was CrkL was diminished ϳ50% compared with WT (Fig. 4B and NS1 of A/Brevig (B), A/Mallard (M), or A/Udorn (U) was data not shown). These data formally establish the critical role included, the IFN-␤-induced increase in reporter gene expres- data not shown). These data formally establish the critical role included, the IFN-␤-induced increase in reporter gene expres- of the class II SH3-binding motif found in A/Brevig and many sion was strongly suppressed. Similar inhibition was also seen of the class II SH3-binding motif found in A/Brevig and many sion was strongly suppressed. Similar inhibition was also seen avian strains of influenza A in Crk/CrkL association, and con- when SH3 binding-deficient mutants (corresponding to M1 in avian strains of influenza A in Crk/CrkL association, and con- when SH3 binding-deficient mutants (corresponding to M1 in firm the preference of Crk/CrkL SH3 domains for lysine-con- Fig. 5A) of A/Brevig (Bm) or A/Mallard (Mm) were tested, which firm the preference of Crk/CrkL SH3 domains for lysine-con- Fig. 5A) of A/Brevig (Bm) or A/Mallard (Mm) were tested, which taining class II-binding sites. together with the activity of A/Udorn in this assay indicated taining class II-binding sites. together with the activity of A/Udorn in this assay indicated Role of SH3 Binding in Cellular Functions of NS1—To study that binding to Crk/CrkL was not required for the capacity of Role of SH3 Binding in Cellular Functions of NS1—To study that binding to Crk/CrkL was not required for the capacity of the functional role and relevance of Crk/CrkL binding by NS1, influenza A NS1 proteins to suppress interferon-induced gene the functional role and relevance of Crk/CrkL binding by NS1, influenza A NS1 proteins to suppress interferon-induced gene we tested the activities of A/Brevig and A/Mallard NS1 proteins expression. we tested the activities of A/Brevig and A/Mallard NS1 proteins expression. and their SH3 binding-deficient derivatives in two different cel- By contrast, SH3 binding by NS1 was found to correlate with and their SH3 binding-deficient derivatives in two different cel- By contrast, SH3 binding by NS1 was found to correlate with lular functions that have been reported for NS1, namely inhibi- a significant increase in Akt phosphorylation in Huh-7 cells lular functions that have been reported for NS1, namely inhibi- a significant increase in Akt phosphorylation in Huh-7 cells tion of interferon-induced gene expression and activation of transfected with these vectors. As noted by others (6), cationic tion of interferon-induced gene expression and activation of transfected with these vectors. As noted by others (6), cationic the PI3K signaling pathway. The former apparently involves lipid-mediated transfection of an empty expression vector plas- the PI3K signaling pathway. The former apparently involves lipid-mediated transfection of an empty expression vector plas- multiple effector functions of NS1, notably the ability of NS1 to mid was sufficient to induce a minor activation of PI3K signal- multiple effector functions of NS1, notably the ability of NS1 to mid was sufficient to induce a minor activation of PI3K signal- interfere with post-transcriptional mRNA processing (38–40). ing. However, in agreement with earlier reports (6, 8), the use of interfere with post-transcriptional mRNA processing (38–40). ing. However, in agreement with earlier reports (6, 8), the use of As already discussed, the latter involves association of NS1 with vectors encoding NS1 of A/Udorn (U) or A/WSN (W) resulted As already discussed, the latter involves association of NS1 with vectors encoding NS1 of A/Udorn (U) or A/WSN (W) resulted

5724 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 9•FEBRUARY 29, 2008 5724 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 9•FEBRUARY 29, 2008 Enhanced PI3K Activation by NS1 via SH3 Binding Enhanced PI3K Activation by NS1 via SH3 Binding in further accumulation of pAkt as an indication of PI3K acti- Indeed, NS1-mediated activation of PI3K signaling was recently in further accumulation of pAkt as an indication of PI3K acti- Indeed, NS1-mediated activation of PI3K signaling was recently vation (see Fig. 6B). By comparison, the levels of pAkt were shown to enhance influenza A virus replication (9, 10) and limit vation (see Fig. 6B). By comparison, the levels of pAkt were shown to enhance influenza A virus replication (9, 10) and limit significantly higher in cells transfected with A/Brevig (B)or apoptosis in infected cell cultures (8). Our observation that a significantly higher in cells transfected with A/Brevig (B)or apoptosis in infected cell cultures (8). Our observation that a A/Mallard (M) NS1 constructs (Fig. 6B and data not shown). functional Crk/CrkL-binding motif provided the NS1 proteins A/Mallard (M) NS1 constructs (Fig. 6B and data not shown). functional Crk/CrkL-binding motif provided the NS1 proteins This increased capacity of A/Brevig and Mallard NS1 to acti- of A/Brevig and A/Mallard with an increased ability to induce This increased capacity of A/Brevig and Mallard NS1 to acti- of A/Brevig and A/Mallard with an increased ability to induce vate PI3K signaling was lost when the class II consensus SH3- PI3K signaling could therefore readily explain a potentially vate PI3K signaling was lost when the class II consensus SH3- PI3K signaling could therefore readily explain a potentially binding motif of these proteins was disrupted by the M1 muta- higher replicative capacity of viruses expressing Crk/CrkL binding motif of these proteins was disrupted by the M1 muta- higher replicative capacity of viruses expressing Crk/CrkL tion (lanes Bm and Mm in Fig. 6B). binding-competent NS1 proteins. tion (lanes Bm and Mm in Fig. 6B). binding-competent NS1 proteins. Crk/CrkL proteins were first identified as cellular counter- Crk/CrkL proteins were first identified as cellular counter- DISCUSSION parts of the protein encoded by the v-crk oncogene of the avian DISCUSSION parts of the protein encoded by the v-crk oncogene of the avian Influenza A viruses are highly virulent and can infect a broad sarcoma virus CT10 (53), and have since been found to be ubiq- Influenza A viruses are highly virulent and can infect a broad sarcoma virus CT10 (53), and have since been found to be ubiq- range of mammalian and avian species. Several influenza A uitously involved in numerous signaling pathways regulating range of mammalian and avian species. Several influenza A uitously involved in numerous signaling pathways regulating virus gene products such as HA, viral polymerases, nucleopro- diverse functions in different cell types (54). Of note, several virus gene products such as HA, viral polymerases, nucleopro- diverse functions in different cell types (54). Of note, several tein, and NS1 play a role in the virulence of the virus (42, 43). studies have reported physical and functional interactions tein, and NS1 play a role in the virulence of the virus (42, 43). studies have reported physical and functional interactions Although the HA protein largely determines the pathogenicity between Crk/CrkL proteins and the PI3K signaling pathway Although the HA protein largely determines the pathogenicity between Crk/CrkL proteins and the PI3K signaling pathway and species specificity of the virus, NS1 protein has a uniform (55–57), thus providing a plausible framework for the mecha- and species specificity of the virus, NS1 protein has a uniform (55–57), thus providing a plausible framework for the mecha- role in regulating host cell responses during the infection inde- nistic basis of the observed enhancement of NS1-mediated role in regulating host cell responses during the infection inde- nistic basis of the observed enhancement of NS1-mediated pendent of the virus type or the animal species the virus is PI3K activation via the NS1-Crk/CrkL interaction. However, pendent of the virus type or the animal species the virus is PI3K activation via the NS1-Crk/CrkL interaction. However, infecting. NS1 protein is a double strand RNA-binding protein, additional roles for the NS1-Crk/CrkL complex in cell biology infecting. NS1 protein is a double strand RNA-binding protein, additional roles for the NS1-Crk/CrkL complex in cell biology and it can interfere with the functions of other double strand of influenza A virus should obviously not be ruled out. and it can interfere with the functions of other double strand of influenza A virus should obviously not be ruled out. RNA-binding proteins such as the RIG-I, protein kinase R, and Previous studies concerning the activation of the PI3K path- RNA-binding proteins such as the RIG-I, protein kinase R, and Previous studies concerning the activation of the PI3K path- oligoadenylate synthetases (44–46) that regulate the induction way by NS1 have shown that this regulation involves an associ- oligoadenylate synthetases (44–46) that regulate the induction way by NS1 have shown that this regulation involves an associ- and antiviral effects of interferons, respectively. NS1 protein is ation of NS1 with the p85 regulatory subunit of PI3K (6, 8, 9), and antiviral effects of interferons, respectively. NS1 protein is ation of NS1 with the p85 regulatory subunit of PI3K (6, 8, 9), also targeted into the host cells nucleus (28), where it can inter- and subsequent studies even further suggested that there is a also targeted into the host cells nucleus (28), where it can inter- and subsequent studies even further suggested that there is a fere with the processing of host cell pre-mRNAs, including direct binding of NS1 to the SH3 domain of p85 (6). However, fere with the processing of host cell pre-mRNAs, including direct binding of NS1 to the SH3 domain of p85 (6). However, those of antiviral mRNAs rendering them susceptible for deg- our studies failed to reveal measurable binding of the SH3 those of antiviral mRNAs rendering them susceptible for deg- our studies failed to reveal measurable binding of the SH3 radation (47). Influenza A virus also takes advantage of the host domains of p85␣ or p85␤ to NS1 proteins like A/Udorn and radation (47). Influenza A virus also takes advantage of the host domains of p85␣ or p85␤ to NS1 proteins like A/Udorn and cell signaling pathways activated during the infection in such a A/WSN, which contain the PXXP sequences pointed out by cell signaling pathways activated during the infection in such a A/WSN, which contain the PXXP sequences pointed out by way that signal transduction involving, for example, NF-␬B, Shin et al. (6) or to A/Brevig or A/Mallard NS1 proteins, which way that signal transduction involving, for example, NF-␬B, Shin et al. (6) or to A/Brevig or A/Mallard NS1 proteins, which mitogen-activated kinase cascades, and the PI3K pathway is contain the additional class II consensus SH3-binding motif mitogen-activated kinase cascades, and the PI3K pathway is contain the additional class II consensus SH3-binding motif altered to optimize virus replication. described in this study. Our results do not exclude the possibil- altered to optimize virus replication. described in this study. Our results do not exclude the possibil- In this study we have shown that the NS1 protein of the 1918 ity that weak binding of p85 SH3 to one of these nonconsensus In this study we have shown that the NS1 protein of the 1918 ity that weak binding of p85 SH3 to one of these nonconsensus pandemic influenza A virus (A/Brevig) contains a functional PXXP motifs in NS1 might play a subtle role in stabilizing or pandemic influenza A virus (A/Brevig) contains a functional PXXP motifs in NS1 might play a subtle role in stabilizing or SH3 interaction motif that mediates avid binding to the N-ter- coordinating NS1/p85 binding, but argue against a major role SH3 interaction motif that mediates avid binding to the N-ter- coordinating NS1/p85 binding, but argue against a major role minal SH3 domain of the adapter proteins Crk and CrkL. This of such contacts in driving NS1-p85 complex formation. minal SH3 domain of the adapter proteins Crk and CrkL. This of such contacts in driving NS1-p85 complex formation. sequence motif is common in NS1 proteins of avian influenza A It should be noted, however, that the ability of recombi- sequence motif is common in NS1 proteins of avian influenza A It should be noted, however, that the ability of recombi- viruses, but besides A/Brevig can be found only in three of the nant p85 SH3 to precipitate NS1 protein from lysates of viruses, but besides A/Brevig can be found only in three of the nant p85 SH3 to precipitate NS1 protein from lysates of 4505 human-derived NS1 protein sequences available in the influenza A-infected cells as reported by Shin et al. (6) does 4505 human-derived NS1 protein sequences available in the influenza A-infected cells as reported by Shin et al. (6) does NCBI influenza A data base. Interestingly, two of these cases not prove this interaction to be direct rather than mediated NCBI influenza A data base. Interestingly, two of these cases not prove this interaction to be direct rather than mediated represent zoonotic infections of humans with avian H5N1 and by one or more additional factors. In fact, it has been represent zoonotic infections of humans with avian H5N1 and by one or more additional factors. In fact, it has been H7N3 viruses. reported that although association of NS1 with p85 can be H7N3 viruses. reported that although association of NS1 with p85 can be Amino acid variation in the NS1 gene has been shown to readily detected in influenza A virus-infected cells, this is Amino acid variation in the NS1 gene has been shown to readily detected in influenza A virus-infected cells, this is correlate with the pathogenicity of H5N1 strains of avian less true in the case of NS1 gene-transfected cells, leading correlate with the pathogenicity of H5N1 strains of avian less true in the case of NS1 gene-transfected cells, leading influenza virus in chickens (48), and the NS1 gene of A/Bre- Ehrhardt et al. (8) to propose that a cellular bridging factor is influenza virus in chickens (48), and the NS1 gene of A/Bre- Ehrhardt et al. (8) to propose that a cellular bridging factor is vig has been found to be more potent than the reference NS1 required for NS1-p85 complex formation. In this regard, it is vig has been found to be more potent than the reference NS1 required for NS1-p85 complex formation. In this regard, it is proteins in regulating host cell gene expression (49). Thus, interesting to note that although we found A/Udorn NS1 to proteins in regulating host cell gene expression (49). Thus, interesting to note that although we found A/Udorn NS1 to although human influenza A infection clearly differs from be completely negative in the CrkL co-precipitation assay in although human influenza A infection clearly differs from be completely negative in the CrkL co-precipitation assay in that of avian hosts in that the capacity of NS1 for Crk/CrkL transfected cells (Fig. 3), we did observe a weak association of that of avian hosts in that the capacity of NS1 for Crk/CrkL transfected cells (Fig. 3), we did observe a weak association of SH3 binding appears to be generally counter-selected in the NS1 and CrkL in cells infected with A/Udorn (data not SH3 binding appears to be generally counter-selected in the NS1 and CrkL in cells infected with A/Udorn (data not formed case, adaptation of the virus to exploit this connec- shown). Thus, in influenza A virus-infected cells expressing formed case, adaptation of the virus to exploit this connec- shown). Thus, in influenza A virus-infected cells expressing tion to the cellular signaling machinery also in human cells high levels of NS1 (as well as other viral proteins that could tion to the cellular signaling machinery also in human cells high levels of NS1 (as well as other viral proteins that could might provide the virus with increased replicative or patho- also be involved) a large protein complex involving NS1, might provide the virus with increased replicative or patho- also be involved) a large protein complex involving NS1, genic potential. It was recently shown that the 1918 virus PI3K, and Crk/CrkL might form independently of direct genic potential. It was recently shown that the 1918 virus PI3K, and Crk/CrkL might form independently of direct shows a higher replication capacity in tissue culture and in binding of NS1 to Crk/CrkL or the p85 subunit of PI3K. shows a higher replication capacity in tissue culture and in binding of NS1 to Crk/CrkL or the p85 subunit of PI3K. animal models (50, 51). However, NS1 proteins like those of A/Brevig and A/Mallard animal models (50, 51). However, NS1 proteins like those of A/Brevig and A/Mallard Several different viral species have been found to stimulate that can directly recruit Crk/CrkL via SH3 binding would Several different viral species have been found to stimulate that can directly recruit Crk/CrkL via SH3 binding would PI3K signaling to increase their replication and/or to prevent serve to promote and strengthen the assembly of this multi- PI3K signaling to increase their replication and/or to prevent serve to promote and strengthen the assembly of this multi- apoptotic death of the host cell (for a review, see Ref. 52). protein “signalosome” complex. apoptotic death of the host cell (for a review, see Ref. 52). protein “signalosome” complex.

FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5725 FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5725 Enhanced PI3K Activation by NS1 via SH3 Binding Enhanced PI3K Activation by NS1 via SH3 Binding

Further characterization of this NS1/PI3K/Crk-containing 20. Longnecker, R., Merchant, M., Brown, M. E., Fruehling, S., Bickford, J. O., Further characterization of this NS1/PI3K/Crk-containing 20. Longnecker, R., Merchant, M., Brown, M. E., Fruehling, S., Bickford, J. O., signalosome is clearly warranted and will shed new light into Ikeda, M., and Harty, R. N. (2000) Exp. Cell Res. 257, 332–340 signalosome is clearly warranted and will shed new light into Ikeda, M., and Harty, R. N. (2000) Exp. Cell Res. 257, 332–340 the cell biology of influenza A replication. Small molecular 21. Bliska, J. (1996) Chem. Biol. 3, 7–11 the cell biology of influenza A replication. Small molecular 21. Bliska, J. (1996) Chem. Biol. 3, 7–11 22. Dupraz, P., Rebai, N., Klein, S. J., Beaulieu, N., and Jolicoeur, P. (1997) 22. Dupraz, P., Rebai, N., Klein, S. J., Beaulieu, N., and Jolicoeur, P. (1997) inhibitors of PI3K are already under evaluation as drugs in J. Virol. 71, 2615–2620 inhibitors of PI3K are already under evaluation as drugs in J. Virol. 71, 2615–2620 cancer (58, 59), and have been shown to reduce influenza A 23. Kay-Jackson, P. C., Goatley, L. C., Cox, L., Miskin, J. E., Parkhouse, R. M., cancer (58, 59), and have been shown to reduce influenza A 23. Kay-Jackson, P. C., Goatley, L. C., Cox, L., Miskin, J. E., Parkhouse, R. M., replication in vitro (7, 9). Thus, cellular signaling involving Wienands, J., and Dixon, L. K. (2004) J. Gen. Virol. 85, 119–130 replication in vitro (7, 9). Thus, cellular signaling involving Wienands, J., and Dixon, L. K. (2004) J. Gen. Virol. 85, 119–130 NS1/PI3K/Crk could also provide a useful target for the 24. Korkaya, H., Jameel, S., Gupta, D., Tyagi, S., Kumar, R., Zafrullah, M., NS1/PI3K/Crk could also provide a useful target for the 24. Korkaya, H., Jameel, S., Gupta, D., Tyagi, S., Kumar, R., Zafrullah, M., development of novel anti-influenza drugs. In this regard, Mazumdar, M., Lal, S. K., Xiaofang, L., Sehgal, D., Das, S. R., and Sahal, D. development of novel anti-influenza drugs. In this regard, Mazumdar, M., Lal, S. K., Xiaofang, L., Sehgal, D., Das, S. R., and Sahal, D. possible involvement of the enhanced PI3K activation medi- (2001) J. Biol. Chem. 276, 42389–42400 possible involvement of the enhanced PI3K activation medi- (2001) J. Biol. Chem. 276, 42389–42400 25. Basler, C. F., Reid, A. H., Dybing, J. K., Janczewski, T. A., Fanning, T. G., 25. Basler, C. F., Reid, A. H., Dybing, J. K., Janczewski, T. A., Fanning, T. G., ated via Crk/CrkL binding by NS1 proteins of avian strains of Zheng, H., Salvatore, M., Perdue, M. L., Swayne, D. E., Garcia-Sastre, A., ated via Crk/CrkL binding by NS1 proteins of avian strains of Zheng, H., Salvatore, M., Perdue, M. L., Swayne, D. E., Garcia-Sastre, A., influenza in regulating interspecies transmission or patho- Palese, P., and Taubenberger, J. K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, influenza in regulating interspecies transmission or patho- Palese, P., and Taubenberger, J. K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, genicity in human hosts deserves special attention. 2746–2751 genicity in human hosts deserves special attention. 2746–2751 26. Obenauer, J. C., Denson, J., Mehta, P. K., Su, X., Mukatira, S., Finkelstein, 26. Obenauer, J. C., Denson, J., Mehta, P. K., Su, X., Mukatira, S., Finkelstein, D. B., Xu, X., Wang, J., Ma, J., Fan, Y., Rakestraw, K. M., Webster, R. G., D. B., Xu, X., Wang, J., Ma, J., Fan, Y., Rakestraw, K. M., Webster, R. G., Acknowledgments—We thank current and past members of the Acknowledgments—We thank current and past members of the Hoffmann, E., Krauss, S., Zheng, J., Zhang, Z., and Naeve, C. W. (2006) Hoffmann, E., Krauss, S., Zheng, J., Zhang, Z., and Naeve, C. W. 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II II

Virology 484 (2015) 146–152 Virology 484 (2015) 146–152

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Virology Virology

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Reorganization of the host cell Crk(L)–PI3 kinase signaling complex Reorganization of the host cell Crk(L)–PI3 kinase signaling complex by the influenza A virus NS1 protein by the influenza A virus NS1 protein

Leena Ylösmäki, Constanze Schmotz, Erkko Ylösmäki, Kalle Saksela n Leena Ylösmäki, Constanze Schmotz, Erkko Ylösmäki, Kalle Saksela n

Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland article info abstract article info abstract

Article history: The non-structural protein-1 (NS1) of influenza A virus binds the p85β subunit of phosphoinositide 3- Article history: The non-structural protein-1 (NS1) of influenza A virus binds the p85β subunit of phosphoinositide 3- Received 27 February 2015 kinase (PI3K) to induce PI3K activity in the infected cells. Some virus strains encode NS1 containing a Received 27 February 2015 kinase (PI3K) to induce PI3K activity in the infected cells. Some virus strains encode NS1 containing a Returned to author for revisions motif that binds tightly to the SH3 domain of the cellular adapter proteins Crk and CrkL to potentiate Returned to author for revisions motif that binds tightly to the SH3 domain of the cellular adapter proteins Crk and CrkL to potentiate 4 June 2015 NS1-induced PI3K activation. Here we show that this potentiation involves reorganization of the natural 4 June 2015 NS1-induced PI3K activation. Here we show that this potentiation involves reorganization of the natural Accepted 5 June 2015 Accepted 5 June 2015 CrkL–p85β complex into a novel trimeric complex where NS1 serves as a bridging factor. Of note, NS1 CrkL–p85β complex into a novel trimeric complex where NS1 serves as a bridging factor. Of note, NS1 Available online 19 June 2015 Available online 19 June 2015 proteins that lack the SH3 binding capacity can also associate with CrkL, but in a less stable trimeric proteins that lack the SH3 binding capacity can also associate with CrkL, but in a less stable trimeric Keywords: complex mediated by p85β. The data presented here establish Crk proteins as general host cell cofactors Keywords: complex mediated by p85β. The data presented here establish Crk proteins as general host cell cofactors fl fl In uenza NS1 protein of NS1, and show that the enhanced PI3K activation by SH3 binding-competent NS1 variants is mediated In uenza NS1 protein of NS1, and show that the enhanced PI3K activation by SH3 binding-competent NS1 variants is mediated SH3 domain SH3 domain by a more efficient tethering of Crk proteins to the NS1–PI3K complex. by a more efficient tethering of Crk proteins to the NS1–PI3K complex. Crk Crk & 2015 Elsevier Inc. All rights reserved. & 2015 Elsevier Inc. All rights reserved. CrkL CrkL PI3K PI3K Signal transduction Signal transduction

Introduction of p85β as well as via direct contacts with the catalytic PI3K Introduction of p85β as well as via direct contacts with the catalytic PI3K subunit p110 (Hale et al., 2008, 2010). subunit p110 (Hale et al., 2008, 2010). Influenza A virus belongs to the Orthomyxoviridae family of Activation of PI3K signaling is not specific for IAV infection, and Influenza A virus belongs to the Orthomyxoviridae family of Activation of PI3K signaling is not specific for IAV infection, and enveloped viruses (Palese and Shaw, 2007). Its genome is orga- instead is a common theme observed in host cell interactions of a enveloped viruses (Palese and Shaw, 2007). Its genome is orga- instead is a common theme observed in host cell interactions of a nized into eight single stranded negative-sense RNA segments and variety of RNA and DNA viruses. Increased PI3K activity can benefit nized into eight single stranded negative-sense RNA segments and variety of RNA and DNA viruses. Increased PI3K activity can benefit encodes up to 12 different proteins (Medina and Garcia-Sastre, viral replication via several mechanisms, including promotion of encodes up to 12 different proteins (Medina and Garcia-Sastre, viral replication via several mechanisms, including promotion of 2011). The non-structural protein-1 (NS1) is a multifunctional viral entry, enhanced viral replication and gene expression, and 2011). The non-structural protein-1 (NS1) is a multifunctional viral entry, enhanced viral replication and gene expression, and virulence factor that is expressed at high levels in infected cells. blocking of premature apoptosis of the host cell (Buchkovich et al., virulence factor that is expressed at high levels in infected cells. blocking of premature apoptosis of the host cell (Buchkovich et al., The multiple protein binding motifs and conformational plasticity 2008; Cooray, 2004; Dunn and Connor, 2012; Ehrhardt and The multiple protein binding motifs and conformational plasticity 2008; Cooray, 2004; Dunn and Connor, 2012; Ehrhardt and of NS1 enables it to interact with a plethora of host cell factors Ludwig, 2009), but has also been shown to be involved in of NS1 enables it to interact with a plethora of host cell factors Ludwig, 2009), but has also been shown to be involved in (Hale, 2014). induction of antiviral innate immunity (Hrincius et al., 2011). (Hale, 2014). induction of antiviral innate immunity (Hrincius et al., 2011). NS1 is the key innate immunity evasion factor of influenza Nevertheless, the net effect of PI3K activity on IAV replication is NS1 is the key innate immunity evasion factor of influenza Nevertheless, the net effect of PI3K activity on IAV replication is viruses, and can hinder the interferon response via multiple typically positive, as demonstrated by reports on profound sup- viruses, and can hinder the interferon response via multiple typically positive, as demonstrated by reports on profound sup- mechanisms (Krug and Garcia-Sastre, 2013). In addition, NS1 pression of viral replication observed upon treatment of infected mechanisms (Krug and Garcia-Sastre, 2013). In addition, NS1 pression of viral replication observed upon treatment of infected contributes to activation of the phosphoinositide 3-kinase (PI3K) cultures with PI3K inhibitors (Ehrhardt et al., 2006; Hale et al., contributes to activation of the phosphoinositide 3-kinase (PI3K) cultures with PI3K inhibitors (Ehrhardt et al., 2006; Hale et al., signaling in influenza A virus-infected cells. NS1 binds specifically 2006). signaling in influenza A virus-infected cells. NS1 binds specifically 2006). to the regulatory PI3K subunit p85β (Hale et al., 2006; Shin et al., While NS1 proteins from most if not all influenza A viruses are to the regulatory PI3K subunit p85β (Hale et al., 2006; Shin et al., While NS1 proteins from most if not all influenza A viruses are 2007) via an interaction that involves the NS1 residues Tyr89 and capable of inducing at least some level of PI3K activation, our 2007) via an interaction that involves the NS1 residues Tyr89 and capable of inducing at least some level of PI3K activation, our Pro164 contacting the linker region between the two Src- studies have revealed that the 1918 pandemic influenza virus and Pro164 contacting the linker region between the two Src- studies have revealed that the 1918 pandemic influenza virus and homology-2 (SH2) domains of p85β (Hale et al., 2010). Together many avian IAVs are exceptionally potent PI3K activators homology-2 (SH2) domains of p85β (Hale et al., 2010). Together many avian IAVs are exceptionally potent PI3K activators biochemical and structural studies on this interaction have led to a (Heikkinen et al., 2008). This distinct capacity was found to be biochemical and structural studies on this interaction have led to a (Heikkinen et al., 2008). This distinct capacity was found to be model in which NS1 activates PI3K catalytic activity both by due to the presence of a functional SH3 domain-binding site close model in which NS1 activates PI3K catalytic activity both by due to the presence of a functional SH3 domain-binding site close neutralizing a negative regulatory element in the inter-SH2 region to the C-termini of these NS1 proteins, which is lacking from NS1 neutralizing a negative regulatory element in the inter-SH2 region to the C-termini of these NS1 proteins, which is lacking from NS1 proteins of influenza A strains causing seasonal epidemics in proteins of influenza A strains causing seasonal epidemics in humans. humans. Although the p85β protein also contains an SH3 domain, we Although the p85β protein also contains an SH3 domain, we

n could not observe any binding of NS1 to this SH3 domain n could not observe any binding of NS1 to this SH3 domain Correspondence to: Department of Virology, University of Helsinki, Haartma- Correspondence to: Department of Virology, University of Helsinki, Haartma- ninkatu 3, PO Box 21, FIN-00014 Helsinki, Finland. Fax: þ358 9 191 26491. (Heikkinen et al., 2008). Instead, we found that the NS1 SH3- ninkatu 3, PO Box 21, FIN-00014 Helsinki, Finland. Fax: þ358 9 191 26491. (Heikkinen et al., 2008). Instead, we found that the NS1 SH3- E-mail address: kalle.saksela@helsinki.fi (K. Saksela). binding motif mediated strong and selective binding to the E-mail address: kalle.saksela@helsinki.fi (K. Saksela). binding motif mediated strong and selective binding to the http://dx.doi.org/10.1016/j.virol.2015.06.009 http://dx.doi.org/10.1016/j.virol.2015.06.009 0042-6822/& 2015 Elsevier Inc. All rights reserved. 0042-6822/& 2015 Elsevier Inc. All rights reserved. L. Ylösmäki et al. / Virology 484 (2015) 146–152 147 L. Ylösmäki et al. / Virology 484 (2015) 146–152 147 aminoterminal SH3 domains of the two related adapter proteins, aminoterminal SH3 domains of the two related adapter proteins, Crk and CrkL (Heikkinen et al., 2008). In the following we use the Crk and CrkL (Heikkinen et al., 2008). In the following we use the term Crk(L) for all Crk-family proteins, and CrkI, CrkII, or CrkL only term Crk(L) for all Crk-family proteins, and CrkI, CrkII, or CrkL only when specifically referring to one of them. SH3-dependent bind- when specifically referring to one of them. SH3-dependent bind- ing of NS1 to Crk(L) has subsequently been confirmed by Ehrhardt ing of NS1 to Crk(L) has subsequently been confirmed by Ehrhardt and colleagues, who also reported on the role of this interaction in and colleagues, who also reported on the role of this interaction in suppression of the JNK–ATF2 pathway (Hrincius et al., 2010). suppression of the JNK–ATF2 pathway (Hrincius et al., 2010). Moreover, a functional Crk(L) binding motif has also been linked Moreover, a functional Crk(L) binding motif has also been linked to NS1-induced increased in cell viability in a complex also to NS1-induced increased in cell viability in a complex also involving NS1-binding protein-1 (Miyazaki et al., 2013), as well involving NS1-binding protein-1 (Miyazaki et al., 2013), as well as in dissociation of cellular Crk–Abl protein complexes and in as in dissociation of cellular Crk–Abl protein complexes and in supression of the tyrosine kinase activity of Abl (Hrincius et al., supression of the tyrosine kinase activity of Abl (Hrincius et al., 2014). 2014). Crk proteins are involved in PI3K signaling by binding to a Crk proteins are involved in PI3K signaling by binding to a proline-rich motif in p85 via their N-terminal Crk(L) SH3 domain proline-rich motif in p85 via their N-terminal Crk(L) SH3 domain (Brehme et al., 2009; Gelkop et al., 2001; Sattler et al., 1996). This (Brehme et al., 2009; Gelkop et al., 2001; Sattler et al., 1996). This interaction has been studied especially regarding its role in PI3K interaction has been studied especially regarding its role in PI3K regulation upon immune cell activation. Studies in T cells have regulation upon immune cell activation. Studies in T cells have shown that the interaction between Crk and p85 is coordinated and shown that the interaction between Crk and p85 is coordinated and facilitated by the ubiquitin ligase Cbl, which can engage in addi- Fig. 1. All Crk proteins have a comparable and SH3 domain-dependent capacity to facilitated by the ubiquitin ligase Cbl, which can engage in addi- Fig. 1. All Crk proteins have a comparable and SH3 domain-dependent capacity to tional interactions both with Crk and p85 (Gelkop et al., 2001). The bind NS1. (a) Domain organization of CrkI, CrkII, and CrkL. (b) Co-precipitation of tional interactions both with Crk and p85 (Gelkop et al., 2001). The bind NS1. (a) Domain organization of CrkI, CrkII, and CrkL. (b) Co-precipitation of Crk–p85–Cbl protein complex has also been characterized as an wild-type (WT) and SH3 binding deficient mutant (AxxA) Mallard NS1 proteins Crk–p85–Cbl protein complex has also been characterized as an wild-type (WT) and SH3 binding deficient mutant (AxxA) Mallard NS1 proteins important co-factor in oncogenic signaling by the Bcr–Abl fusion with CrkI, CrkII, and CrkL from 293T cells. Crk proteins fused with a biotinylation important co-factor in oncogenic signaling by the Bcr–Abl fusion with CrkI, CrkII, and CrkL from 293T cells. Crk proteins fused with a biotinylation domain were cotransfected into cells with myc-tagged NS1 proteins. The amounts domain were cotransfected into cells with myc-tagged NS1 proteins. The amounts protein in chronic myeloid leukemia (Sattler et al., 1996). Indeed, an protein in chronic myeloid leukemia (Sattler et al., 1996). Indeed, an of NS1 in the transfected lysates (bottom panel) or co-precipitated with Crk of NS1 in the transfected lysates (bottom panel) or co-precipitated with Crk extensive proteomic characterization of the molecular network proteins by streptavidin-coated beads (middle panel) were examined by anti-Myc extensive proteomic characterization of the molecular network proteins by streptavidin-coated beads (middle panel) were examined by anti-Myc involved in Bcr–Abl function by Superti-Furga and colleagues Western blotting. Equal precipitation of the biotinylated Crk proteins was con- involved in Bcr–Abl function by Superti-Furga and colleagues Western blotting. Equal precipitation of the biotinylated Crk proteins was con- identified Crk, p85, and Cbl among the seven most highly inter- firmed by blotting with labeled streptavidin (top panel). identified Crk, p85, and Cbl among the seven most highly inter- firmed by blotting with labeled streptavidin (top panel). connected core components of this signaling network (Brehme et connected core components of this signaling network (Brehme et al., 2009). al., 2009). In order to elucidate the biochemical mechanism underlying Relative roles of p85β and Crk-SH3 binding in PI3K activation by NS1 In order to elucidate the biochemical mechanism underlying Relative roles of p85β and Crk-SH3 binding in PI3K activation by NS1 the PI3K superactivation by the SH3 binding-competent NS1 the PI3K superactivation by the SH3 binding-competent NS1 proteins we now have characterized in detail the mutual interac- Hale and colleagues reported the critical contribution of the proteins we now have characterized in detail the mutual interac- Hale and colleagues reported the critical contribution of the tion between NS1, p85β, and CrkL. Because both NS1 and p85β tyrosine 89 of NS1 in direct binding to the regulatory PI3K subunit tion between NS1, p85β, and CrkL. Because both NS1 and p85β tyrosine 89 of NS1 in direct binding to the regulatory PI3K subunit interact with the Crk proteins via the same N-terminal Crk(L) SH3 p85β (Hale et al., 2006). On the other hand, we have shown that interact with the Crk proteins via the same N-terminal Crk(L) SH3 p85β (Hale et al., 2006). On the other hand, we have shown that domain it was of specific interest to investigate whether a trimeric NS1 containing a functional Crk-SH3 binding site show greatly domain it was of specific interest to investigate whether a trimeric NS1 containing a functional Crk-SH3 binding site show greatly complex involving p85β, Crk(L) and NS1 can form. enhanced PI3K activation compared to SH3 binding-deficient NS1 complex involving p85β, Crk(L) and NS1 can form. enhanced PI3K activation compared to SH3 binding-deficient NS1 (Heikkinen et al., 2008). To investigate if the increased PI3K (Heikkinen et al., 2008). To investigate if the increased PI3K activation provided by NS1 SH3 binding was directly coupled to activation provided by NS1 SH3 binding was directly coupled to the NS1–p85β interaction or represented an independent regula- the NS1–p85β interaction or represented an independent regula- Results tory event, we compared the effects of the AxxA (P212A/P215A; Results tory event, we compared the effects of the AxxA (P212A/P215A; disrupting Crk SH3 binding) and Y89F (disrupting p85β binding) disrupting Crk SH3 binding) and Y89F (disrupting p85β binding) Association of different Crk proteins with NS1 and p85β mutations. Association of different Crk proteins with NS1 and p85β mutations. In agreement with our earlier study (Heikkinen et al., 2008)we In agreement with our earlier study (Heikkinen et al., 2008)we The family of Crk adapter proteins includes three members, found that the PI3K-activating capacity of Mallard NS1 (induction The family of Crk adapter proteins includes three members, found that the PI3K-activating capacity of Mallard NS1 (induction CrkI, CrkII, and CrkL. Of these CrkII (40 kDa) and CrkI (28 kDa) are of Akt phosphorylation) was reduced but not abrogated by the CrkI, CrkII, and CrkL. Of these CrkII (40 kDa) and CrkI (28 kDa) are of Akt phosphorylation) was reduced but not abrogated by the products of the same gene, whereas CrkL (39 kDa) is encoded by a AxxA mutation (Fig. 2a). By contrast, the Y89F mutant NS1 was products of the same gene, whereas CrkL (39 kDa) is encoded by a AxxA mutation (Fig. 2a). By contrast, the Y89F mutant NS1 was different gene. CrkII and CrkL consist of one SH2 and two SH3 completely inactive in inducing Akt phosphorylation. Thus, we different gene. CrkII and CrkL consist of one SH2 and two SH3 completely inactive in inducing Akt phosphorylation. Thus, we domains, while CrkI is truncated before the second SH3 domain conclude that Crk(L) recruitment by NS1 indeed serves to enhance domains, while CrkI is truncated before the second SH3 domain conclude that Crk(L) recruitment by NS1 indeed serves to enhance due to alternative mRNA splicing (Fig. 1a). PI3K activation triggered by binding of NS1 to p85β, but this due to alternative mRNA splicing (Fig. 1a). PI3K activation triggered by binding of NS1 to p85β, but this To extend our previous findings on NS1/Crk(L) binding enhancement remains entirely dependent on the latter To extend our previous findings on NS1/Crk(L) binding enhancement remains entirely dependent on the latter (Heikkinen et al., 2008), we compared the capacity of these three interaction. (Heikkinen et al., 2008), we compared the capacity of these three interaction. different Crk proteins to interact with a representative avian NS1 different Crk proteins to interact with a representative avian NS1 protein containing a functional SH3 binding site (NS1 from A/ Rearrangement of the endogenous p85β/CrkL complex by NS1 protein containing a functional SH3 binding site (NS1 from A/ Rearrangement of the endogenous p85β/CrkL complex by NS1 Mallard/Netherlands/12/2000/H7N3; “Mallard” in the following). Mallard/Netherlands/12/2000/H7N3; “Mallard” in the following). To examine the role of NS1 SH3 binding, we also included in these Because p85 binds not only to NS1 (Hale et al., 2006; Shin et al., To examine the role of NS1 SH3 binding, we also included in these Because p85 binds not only to NS1 (Hale et al., 2006; Shin et al., experiments a mutated version of Mallard NS1 protein (NS1- 2007) but is also an established interaction partner of the Crk experiments a mutated version of Mallard NS1 protein (NS1- 2007) but is also an established interaction partner of the Crk AxxA), carrying an SH3 binding site disrupted by alanine substitu- proteins (Brehme et al., 2009; Sattler et al., 1996, 1997)we AxxA), carrying an SH3 binding site disrupted by alanine substitu- proteins (Brehme et al., 2009; Sattler et al., 1996, 1997)we tion of two critical proline residues (P212 and P215). As shown in examined the association of p85β with CrkL in untransfected cells tion of two critical proline residues (P212 and P215). As shown in examined the association of p85β with CrkL in untransfected cells Fig. 1b, NS1 associated readily with CrkI, CrkII, as well as CrkL, and in cells transfected with the mutant NS1 proteins. Indeed, Fig. 1b, NS1 associated readily with CrkI, CrkII, as well as CrkL, and in cells transfected with the mutant NS1 proteins. Indeed, correlating with the presence of an N-terminal SH3 domain in all p85β could be readily co-precipitated with CrkL from untrans- correlating with the presence of an N-terminal SH3 domain in all p85β could be readily co-precipitated with CrkL from untrans- of these Crk proteins (Fig. 1a). In contrast, none of the Crk proteins fected cells that did not express any NS1 (Fig. 2b, leftmost lane). of these Crk proteins (Fig. 1a). In contrast, none of the Crk proteins fected cells that did not express any NS1 (Fig. 2b, leftmost lane). co-precipitated the SH3 binding-deficient NS1-AxxA. The blotting Transfection of the CrkL binding-competent (WT) or incompetent co-precipitated the SH3 binding-deficient NS1-AxxA. The blotting Transfection of the CrkL binding-competent (WT) or incompetent data shown in Fig. 1b are representative of several experiments, (AxxA) NS1 proteins did not significantly influence the net data shown in Fig. 1b are representative of several experiments, (AxxA) NS1 proteins did not significantly influence the net and all results shown have been reproduced at least three times. association of p85β with CrkL. However, transfection of the Y89F and all results shown have been reproduced at least three times. association of p85β with CrkL. However, transfection of the Y89F The same holds true for the blotting data shown in Figs. 2, 4, 5 and mutant of NS1 (which is CrkL binding-competent but p85β The same holds true for the blotting data shown in Figs. 2, 4, 5 and mutant of NS1 (which is CrkL binding-competent but p85β 6. binding-deficient) potently inhibited p85β/CrkL association. Since 6. binding-deficient) potently inhibited p85β/CrkL association. Since 148 L. Ylösmäki et al. / Virology 484 (2015) 146–152 148 L. Ylösmäki et al. / Virology 484 (2015) 146–152 p85β and NS1 compete for the same N-terminal SH3 domain of p85β and NS1 compete for the same N-terminal SH3 domain of CrkL, this observation suggested that NS1 could displace p85β CrkL, this observation suggested that NS1 could displace p85β from binding to CrkL due to a higher affinity for the CrkL SH3 from binding to CrkL due to a higher affinity for the CrkL SH3 domain. domain. To directly demonstrate that NS1 could displace p85β from a To directly demonstrate that NS1 could displace p85β from a preformed p85β–CrkL complex, we co-transfected cells with CrkL preformed p85β–CrkL complex, we co-transfected cells with CrkL and p85β without NS1, and added increasing amounts of recom- and p85β without NS1, and added increasing amounts of recom- binant NS1–Y89F into a lysate of these cells to examine whether binant NS1–Y89F into a lysate of these cells to examine whether p85β or NS1 co-precipitated with CrkL. As shown in Fig. 2c p85β or NS1 co-precipitated with CrkL. As shown in Fig. 2c recombinant NS1–Y89F displaced p85β from its complex with recombinant NS1–Y89F displaced p85β from its complex with CrkL in a dose-dependent manner. The highest dose (12 μg) of CrkL in a dose-dependent manner. The highest dose (12 μg) of NS1–Y89F (corresponding to a 4-fold molar excess of p85β in the NS1–Y89F (corresponding to a 4-fold molar excess of p85β in the lysate) completely abrogated association of p85β with CrkL. By lysate) completely abrogated association of p85β with CrkL. By contrast, 12 μg of NS1-AxxA had no effect. As could be predicted, a contrast, 12 μg of NS1-AxxA had no effect. As could be predicted, a similar displacement of 85β from the complex with CrkL by wild- similar displacement of 85β from the complex with CrkL by wild- type NS1 could be also demonstrated when NS1–p85β binding type NS1 could be also demonstrated when NS1–p85β binding

Fig. 3. Reported (a) and hypothesized (b) interactions among the Crk(L), p85β, and Fig. 3. Reported (a) and hypothesized (b) interactions among the Crk(L), p85β, and NS1 proteins. Note that all natural NS1 proteins do not contain the SH3 target motif NS1 proteins. Note that all natural NS1 proteins do not contain the SH3 target motif (PxxP) formed by the NS1 residues P212 and P215. Hence this motif has been (PxxP) formed by the NS1 residues P212 and P215. Hence this motif has been drawn only when it is necessary for the indicated complex. As discussed in the text, drawn only when it is necessary for the indicated complex. As discussed in the text, the affinity of the Crk(L) SH3 domain for the NS1 PxxP motif appears to be greater the affinity of the Crk(L) SH3 domain for the NS1 PxxP motif appears to be greater than its affinity for the p85β PxxP motif. The presence or absence of this motif in than its affinity for the p85β PxxP motif. The presence or absence of this motif in NS1 may therefore determine which of the two alternative trimeric complexes NS1 may therefore determine which of the two alternative trimeric complexes illustrated in (b) is formed. The references for the previously reported dimeric illustrated in (b) is formed. The references for the previously reported dimeric complexes shown in (a) are Crk(L)–p85β (Gelkop et al., 2001; Sattler et al., 1997), complexes shown in (a) are Crk(L)–p85β (Gelkop et al., 2001; Sattler et al., 1997), NS1–p85β (Hale et al., 2006; Shin et al., 2007), and in (b) NS1–Crk(L) (Heikkinen et NS1–p85β (Hale et al., 2006; Shin et al., 2007), and in (b) NS1–Crk(L) (Heikkinen et al., 2008). al., 2008).

was prevented by specific mutations in p85β (p85β-V573M) was prevented by specific mutations in p85β (p85β-V573M) (Supplementary Fig. 1). (Supplementary Fig. 1). These results suggested that co-precipitation of p85β with CrkL These results suggested that co-precipitation of p85β with CrkL observed in cells expressing wild-type NS1 (Fig. 2b) was probably observed in cells expressing wild-type NS1 (Fig. 2b) was probably not direct, but instead indirectly mediated via the dual capacity of not direct, but instead indirectly mediated via the dual capacity of NS1 to bind both CrkL and p85β, thus indicating a scenario where NS1 to bind both CrkL and p85β, thus indicating a scenario where the NS1-mediated enhancement of PI3K activation would involve the NS1-mediated enhancement of PI3K activation would involve a rearrangement of the natural CrkL–p85β complex and lead to a rearrangement of the natural CrkL–p85β complex and lead to formation of a trimeric CrkL–NS1–p85β complex (where NS1 is formation of a trimeric CrkL–NS1–p85β complex (where NS1 is the bridging factor; see Fig. 3). Assembly of such a trimeric the bridging factor; see Fig. 3). Assembly of such a trimeric complex (as opposed to separate CrkL–NS1 and NS1–p85β com- complex (as opposed to separate CrkL–NS1 and NS1–p85β com- plexes) is enabled by the abundant cellular expression of CrkL. plexes) is enabled by the abundant cellular expression of CrkL. On the other hand, based on the capacity of Crk(L), p85, and On the other hand, based on the capacity of Crk(L), p85, and NS1 to form dimeric complexes (see Fig. 3a), one could hypothe- NS1 to form dimeric complexes (see Fig. 3a), one could hypothe- size that the complex of SH3 binding-deficient NS1 with p85β size that the complex of SH3 binding-deficient NS1 with p85β should also contain CrkL, thus representing an alternative trimeric should also contain CrkL, thus representing an alternative trimeric CrkL–p85β–NS1 complex (where p85β is the bridging factor). CrkL–p85β–NS1 complex (where p85β is the bridging factor). Seemingly in conflict with this assumption, only the NS1 Seemingly in conflict with this assumption, only the NS1 proteins capable of direct CrkL binding (WT and Y89F) but no proteins capable of direct CrkL binding (WT and Y89F) but no

Fig. 2. Mutational analysis of the roles of SH3- and p85β-binding by NS1. NS1-AxxA were found in anti-CrkL immunocomplexes (Fig. 2b). Fig. 2. Mutational analysis of the roles of SH3- and p85β-binding by NS1. NS1-AxxA were found in anti-CrkL immunocomplexes (Fig. 2b). (a) Activation of PI3K signaling by wild-type (WT), SH3-binding deficient (AxxA), Since this could be explained by a modest affinity of the CrkL SH3 (a) Activation of PI3K signaling by wild-type (WT), SH3-binding deficient (AxxA), Since this could be explained by a modest affinity of the CrkL SH3 or p85β-binding deficient (Y89F) NS1 proteins. Myc-tagged versions of the domain for p85β and/or a low abundance of p85β compared to or p85β-binding deficient (Y89F) NS1 proteins. Myc-tagged versions of the domain for p85β and/or a low abundance of p85β compared to indicated NS1 proteins were transfected into Huh7 cells. 48 h later activation of CrkL and NS1, we examined if overexpression of p85β would allow indicated NS1 proteins were transfected into Huh7 cells. 48 h later activation of CrkL and NS1, we examined if overexpression of p85β would allow PI3K signaling was assayed by Western blotting of lysates of the transfected cells PI3K signaling was assayed by Western blotting of lysates of the transfected cells us to detect p85β-mediated bridging of CrkL and NS1-AxxA. Thus, us to detect p85β-mediated bridging of CrkL and NS1-AxxA. Thus, with an antibody specific for phospho-Akt (top panel). Equal amounts of the with an antibody specific for phospho-Akt (top panel). Equal amounts of the different NS1 proteins in these lysates were confirmed by anti-Myc Western we co-transfected increasing amounts of p85β into cells together different NS1 proteins in these lysates were confirmed by anti-Myc Western we co-transfected increasing amounts of p85β into cells together blotting (middle panel). (b) Co-precipitation of endogenous p85β and transfected with a constant amount of wild-type NS1 or NS1-AxxA. As blotting (middle panel). (b) Co-precipitation of endogenous p85β and transfected with a constant amount of wild-type NS1 or NS1-AxxA. As NS1 proteins (WT, AxxA, or Y89F) with endogenous CrkL. Lysates of cells expected, coprecipitation of wild-type NS1 with CrkL was already NS1 proteins (WT, AxxA, or Y89F) with endogenous CrkL. Lysates of cells expected, coprecipitation of wild-type NS1 with CrkL was already transfected as in A were subjected to anti-CrkL immunoprecipitation followed by fi β transfected as in A were subjected to anti-CrkL immunoprecipitation followed by fi β β ef cient in the absence of p85 transfection, and did not sig- β ef cient in the absence of p85 transfection, and did not sig- Western blotting analysis of the immunocomplexes with an anti-p85 (middle fi β Western blotting analysis of the immunocomplexes with an anti-p85 (middle fi β panel) or an anti-Myc (NS1) antibody (bottom panel). Equal NS1 expression in the ni cantly change upon ectopic p85 expression (Fig. 4a). By panel) or an anti-Myc (NS1) antibody (bottom panel). Equal NS1 expression in the ni cantly change upon ectopic p85 expression (Fig. 4a). By total lysates was confirmed as in (a). (c) Competitive disruption of the CrkL–p85β contrast, increasing amounts of p85β enabled coprecipitation of total lysates was confirmed as in (a). (c) Competitive disruption of the CrkL–p85β contrast, increasing amounts of p85β enabled coprecipitation of complex by titration of recombinant NS1–Y89F. Increasing amounts (from 0 μgto NS1-AxxA with CrkL in a dose-dependent manner, resulting in complex by titration of recombinant NS1–Y89F. Increasing amounts (from 0 μgto NS1-AxxA with CrkL in a dose-dependent manner, resulting in 12 μg) of recombinant NS1–Y89F or 12 μg of NS1-AxxA expressed as GST fusion CrkL-association almost as efficient as observed for wild-type NS1. 12 μg) of recombinant NS1–Y89F or 12 μg of NS1-AxxA expressed as GST fusion CrkL-association almost as efficient as observed for wild-type NS1. proteins were added to lysates of cells transfected with biotinylation domain- proteins were added to lysates of cells transfected with biotinylation domain- Thus, we concluded that a p85β-mediated CrkL–p85β–NS1 com- Thus, we concluded that a p85β-mediated CrkL–p85β–NS1 com- tagged CrkL and HA-tagged p85β (total protein amount 200 μg). The amount of tagged CrkL and HA-tagged p85β (total protein amount 200 μg). The amount of p85β and NS1 proteins associated with CrkL precipitated with streptavidin beads plex does form in cells expressing an SH3 binding-incompetent p85β and NS1 proteins associated with CrkL precipitated with streptavidin beads plex does form in cells expressing an SH3 binding-incompetent was examined by Western blotting. NS1 protein. was examined by Western blotting. NS1 protein. L. Ylösmäki et al. / Virology 484 (2015) 146–152 149 L. Ylösmäki et al. / Virology 484 (2015) 146–152 149

Demonstration of a p85β-bridged association of CrkL and the Demonstration of a p85β-bridged association of CrkL and the Mallard NS1-AxxA suggested that similar trimeric Crk(L)–p85β– Mallard NS1-AxxA suggested that similar trimeric Crk(L)–p85β– NS1 complexes would also be formed in cells expressing NS1 NS1 complexes would also be formed in cells expressing NS1 proteins encoded by prototypic viruses, such as A/WSN/33 and A/ proteins encoded by prototypic viruses, such as A/WSN/33 and A/ Udorn/72, which represent seasonal human IAV strains lacking the Udorn/72, which represent seasonal human IAV strains lacking the Crk(L) SH3 binding motif. Indeed, when WSN and Udorn NS1 Crk(L) SH3 binding motif. Indeed, when WSN and Udorn NS1 proteins were co-transfected with p85β, they could be co- proteins were co-transfected with p85β, they could be co- precipitated with cellular CrkL as efficiently as the Mallard NS1 precipitated with cellular CrkL as efficiently as the Mallard NS1 protein (Fig. 4b). In strong support of the model of the two protein (Fig. 4b). In strong support of the model of the two alternative CrkL–p85β–NS1 complexes (Fig. 3b), the capacity of alternative CrkL–p85β–NS1 complexes (Fig. 3b), the capacity of p85β to couple Udorn and WSN NS1 with CrkL completely p85β to couple Udorn and WSN NS1 with CrkL completely depended on the SH3 binding motif of p85β, and was disrupted depended on the SH3 binding motif of p85β, and was disrupted by the P294A/P297A mutation. Thus, we conclude that Crk by the P294A/P297A mutation. Thus, we conclude that Crk proteins are also associated with the PI3K-activating NS1/p85β proteins are also associated with the PI3K-activating NS1/p85β complexes involving NS1 proteins that lack direct Crk(L) binding complexes involving NS1 proteins that lack direct Crk(L) binding capacity. capacity. To further test the model of two alternative trimeric complexes To further test the model of two alternative trimeric complexes (Fig. 3b), we co-expressed all relevant combinations of the (Fig. 3b), we co-expressed all relevant combinations of the differentially mutated versions of these proteins, and investigated Fig. 5. Mutational characterization of the interaction interfaces involved in the differentially mutated versions of these proteins, and investigated Fig. 5. Mutational characterization of the interaction interfaces involved in the in parallel which complexes could form (Fig. 5). For these studies assembly of two alternative trimeric complexes between CrkL, NS1, and p85β. in parallel which complexes could form (Fig. 5). For these studies assembly of two alternative trimeric complexes between CrkL, NS1, and p85β. β β we generated an additional mutant of p85β (V573M), which Different combinations of wild-type and mutated versions of Mallard NS1 and p85 we generated an additional mutant of p85β (V573M), which Different combinations of wild-type and mutated versions of Mallard NS1 and p85 β were transfected into 293T cells as indicated. Immunocomplexes precipitated from β were transfected into 293T cells as indicated. Immunocomplexes precipitated from prevents the binding of p85 to NS1 (Li et al., 2008). As already these cells by antibodies against CrkL (endogenous) (panels I-III) or p85β (anti-HA; prevents the binding of p85 to NS1 (Li et al., 2008). As already these cells by antibodies against CrkL (endogenous) (panels I-III) or p85β (anti-HA; shown for endogenous p85β (Fig. 2b), transfected wild-type p85β- panels IV-VI) were examined by probing the Western blot with antibodies against shown for endogenous p85β (Fig. 2b), transfected wild-type p85β- panels IV-VI) were examined by probing the Western blot with antibodies against HA could be co-precipitated with CrkL from wild-type NS1- and p85β (anti-HA), NS1, or CrkL as indicated. Expression of NS1 in all NS1-transfected HA could be co-precipitated with CrkL from wild-type NS1- and p85β (anti-HA), NS1, or CrkL as indicated. Expression of NS1 in all NS1-transfected cells was confirmed by Western blot analysis of total cell lysates (WCE). cells was confirmed by Western blot analysis of total cell lysates (WCE).

NS1-AxxA-transfected cells (Fig. 5, panel II, lanes 3 and 4), as well NS1-AxxA-transfected cells (Fig. 5, panel II, lanes 3 and 4), as well as in the absence of any NS1 (lane 2), whereas expression of NS1– as in the absence of any NS1 (lane 2), whereas expression of NS1– Y89F inhibited this association (lane 5). In the absence of NS1 the Y89F inhibited this association (lane 5). In the absence of NS1 the V573M mutation in p85β did not affect binding to CrkL (lane 10), V573M mutation in p85β did not affect binding to CrkL (lane 10), but this mutation rendered p85β–CrkL association sensitive to but this mutation rendered p85β–CrkL association sensitive to inhibition also by wild-type NS1 (lane 11). In keeping with the inhibition also by wild-type NS1 (lane 11). In keeping with the earlier work (Gelkop et al., 2001; Sattler et al., 1997), disruption of earlier work (Gelkop et al., 2001; Sattler et al., 1997), disruption of the SH3 binding site in p85β (P294A, P297A) abolished direct the SH3 binding site in p85β (P294A, P297A) abolished direct binding of p85β to CrkL (lane 6). Attesting to the role of NS1 as a binding of p85β to CrkL (lane 6). Attesting to the role of NS1 as a bridging factor between CrkL and p85β in Complex 1 (Fig. 3b) co- bridging factor between CrkL and p85β in Complex 1 (Fig. 3b) co- precipitation of p85β-P294A, P297A with CrkL could be rescued by precipitation of p85β-P294A, P297A with CrkL could be rescued by wild-type NS1 (Fig. 5, lane 7), but not by NS1-AxxA or NS1-Y89F wild-type NS1 (Fig. 5, lane 7), but not by NS1-AxxA or NS1-Y89F (lanes 8 and 9). The same was true for co-precipitation of CrkL (lanes 8 and 9). The same was true for co-precipitation of CrkL with p85β (panel VI). Only wild-type NS1 could mediate pull- with p85β (panel VI). Only wild-type NS1 could mediate pull- down of CrkL by p85β–P294A, P297A, whereas co-precipitation of down of CrkL by p85β–P294A, P297A, whereas co-precipitation of CrkL by wild-type p85β was observed even in the absence of any CrkL by wild-type p85β was observed even in the absence of any NS1. Of note, however, wild-type NS1 further increased CrkL/ NS1. Of note, however, wild-type NS1 further increased CrkL/ p85β-association, attesting to the efficient formation of a trimeric p85β-association, attesting to the efficient formation of a trimeric CrkL–NS1–p85β complex. This effect also evident in the reciprocal CrkL–NS1–p85β complex. This effect also evident in the reciprocal co-IP (panel II), albeit less pronounced presumably because of the co-IP (panel II), albeit less pronounced presumably because of the higher relative abundance of transfected p85β compared to higher relative abundance of transfected p85β compared to endogenous CrkL. endogenous CrkL. In addition to the expected coprecipitation pattern of the p85β In addition to the expected coprecipitation pattern of the p85β variants, the presence of NS1 and its AxxA and Y89F mutants in variants, the presence of NS1 and its AxxA and Y89F mutants in the anti-CrkL (panel III) or in the anti-p85β (anti-HA; panel V) the anti-CrkL (panel III) or in the anti-p85β (anti-HA; panel V) immunocomplexes correlated perfectly with the model presented immunocomplexes correlated perfectly with the model presented in Fig. 3(b). Specifically, V573M mutation in p85β or Y89F muta- in Fig. 3(b). Specifically, V573M mutation in p85β or Y89F muta- tion in NS1 prevented interaction of NS1 with p85β, while SH3 tion in NS1 prevented interaction of NS1 with p85β, while SH3 binding capacity of NS1 did not play a role. In contrast, whereas binding capacity of NS1 did not play a role. In contrast, whereas the SH3 binding-competent NS1-WT and NS1–Y89F proteins could the SH3 binding-competent NS1-WT and NS1–Y89F proteins could be precipitated with CrkL regardless of the cotransfected p85β be precipitated with CrkL regardless of the cotransfected p85β Fig. 4. Overexpression of p85β reveals association of CrkL with NS1/p85β com- variant, NS1-AxxA failed to associate with CrkL if either the SH3 Fig. 4. Overexpression of p85β reveals association of CrkL with NS1/p85β com- variant, NS1-AxxA failed to associate with CrkL if either the SH3 plexes involving SH3 binding-incompetent NS1 proteins. (a) Increasing amounts of binding site (P294A, P297A) or the NS1 binding site (V573M) of plexes involving SH3 binding-incompetent NS1 proteins. (a) Increasing amounts of binding site (P294A, P297A) or the NS1 binding site (V573M) of HA-tagged p85β was transfected to 293T cells together with wild-type (WT) or β HA-tagged p85β was transfected to 293T cells together with wild-type (WT) or β SH3-binding deficient (AxxA) NS1. Proteins associated with anti-CrkL (endogenous) p85 was mutated. SH3-binding deficient (AxxA) NS1. Proteins associated with anti-CrkL (endogenous) p85 was mutated. precipitations from these cells were detected in Western blots with anti-HA (p85β), To confirm that such trimolecular complexes can assemble as precipitations from these cells were detected in Western blots with anti-HA (p85β), To confirm that such trimolecular complexes can assemble as anti-CrkL, or anti-NS1 antibodies. The levels of transfected p85β and NS1 in the depicted in Fig. 3b without the assistance of additional CrkL- or anti-CrkL, or anti-NS1 antibodies. The levels of transfected p85β and NS1 in the depicted in Fig. 3b without the assistance of additional CrkL- or total lysates (WCE) were similarly detected. (b) NS1 proteins from Mallard (M), p85β-associated cellular factors, we expressed and purified full- total lysates (WCE) were similarly detected. (b) NS1 proteins from Mallard (M), p85β-associated cellular factors, we expressed and purified full- WSN (W), or Udorn (U) strains were cotransfected to 293T cells together with a WSN (W), or Udorn (U) strains were cotransfected to 293T cells together with a length p85β (both wild-type and the P294A/P297A mutant) and length p85β (both wild-type and the P294A/P297A mutant) and wild-type (WT) or Crk(L) SH3 domain binding-deficient mutant (P294A, P297A) wild-type (WT) or Crk(L) SH3 domain binding-deficient mutant (P294A, P297A) versions of p85β. The association of these NS1 with anti-CrkL immunocomplexes NS1 (both wild-type and the AxxA mutant) proteins, as well as a versions of p85β. The association of these NS1 with anti-CrkL immunocomplexes NS1 (both wild-type and the AxxA mutant) proteins, as well as a was examined as in (a). CrkL protein fragment containing the SH3 domain in recombinant was examined as in (a). CrkL protein fragment containing the SH3 domain in recombinant 150 L. Ylösmäki et al. / Virology 484 (2015) 146–152 150 L. Ylösmäki et al. / Virology 484 (2015) 146–152 form. Experiments involving mixing of two or three of these form. Experiments involving mixing of two or three of these proteins together followed by pull-down analysis of proteins proteins together followed by pull-down analysis of proteins associated with wild-type or mutant p85β confirmed that Com- associated with wild-type or mutant p85β confirmed that Com- plex I (where NS1 connects p85β and CrkL) as well as Complex II plex I (where NS1 connects p85β and CrkL) as well as Complex II (where p85β connects NS1 and CrkL) can be assembled in vitro (where p85β connects NS1 and CrkL) can be assembled in vitro from purified recombinant components (Supplementary Fig. 2). from purified recombinant components (Supplementary Fig. 2). Moreover, confirming our conclusions on the assembly of these Moreover, confirming our conclusions on the assembly of these complexes in living cells, SH3 binding-deficient NS1 could associ- complexes in living cells, SH3 binding-deficient NS1 could associ- ate with CrkL in Complex II only if the SH3 binding site in p85β ate with CrkL in Complex II only if the SH3 binding site in p85β was intact. was intact. In conclusion, these data firmly established a model where a In conclusion, these data firmly established a model where a trimeric complex involving NS1, CrkL, and p85β can assemble trimeric complex involving NS1, CrkL, and p85β can assemble according to two distinct principles, depending on the SH3 binding according to two distinct principles, depending on the SH3 binding capacity of NS1 (Fig. 3b). capacity of NS1 (Fig. 3b).

Functional consequences of p85β/CrkL complex rearrangement by Functional consequences of p85β/CrkL complex rearrangement by NS1 NS1

As already discussed, Crk proteins are naturally associated with As already discussed, Crk proteins are naturally associated with PI3K via SH3-mediated binding to a proline-rich motif in p85β. PI3K via SH3-mediated binding to a proline-rich motif in p85β. Because of this interaction recruitment of CrkL to NS1–p85β Because of this interaction recruitment of CrkL to NS1–p85β complexes involving SH3 binding defective NS1 proteins was also complexes involving SH3 binding defective NS1 proteins was also observed (Figs. 4 and 5). However, we could detect a p85β-bridged Fig. 6. Effect of CrkL overexpression on PI3K/Akt activation induced by various NS1 observed (Figs. 4 and 5). However, we could detect a p85β-bridged Fig. 6. Effect of CrkL overexpression on PI3K/Akt activation induced by various NS1 CrkL–NS1 association (Complex 2) only when p85β was over- proteins. (a) Wild-type Mallard NS1 (WT), NS1-AxxA (AxxA), Udorn NS1 (Udorn) or CrkL–NS1 association (Complex 2) only when p85β was over- proteins. (a) Wild-type Mallard NS1 (WT), NS1-AxxA (AxxA), Udorn NS1 (Udorn) or expressed, suggesting that CrkL recruitment to PI3K–NS1 com- an empty control vector (vector) were transfected into Huh7 cells with or without expressed, suggesting that CrkL recruitment to PI3K–NS1 com- an empty control vector (vector) were transfected into Huh7 cells with or without β fi – an expression vector for biotinylation domain-tagged CrkL. Induced PI3K/Akt β fi – an expression vector for biotinylation domain-tagged CrkL. Induced PI3K/Akt plexes via p85 is less ef cient than recruitment via an NS1 CrkL signaling in the transfected cells was examined by probing the lysates with an plexes via p85 is less ef cient than recruitment via an NS1 CrkL signaling in the transfected cells was examined by probing the lysates with an SH3 interaction. Thus, we reasoned that if the lower PI3K activa- antibody specific for phospho-Akt (top panel). The presence of NS1 and CrkL SH3 interaction. Thus, we reasoned that if the lower PI3K activa- antibody specific for phospho-Akt (top panel). The presence of NS1 and CrkL tion by SH3 binding-deficient NS1 proteins might be rescued by proteins in these lysates was detected with anti-Myc antibody or labeled strepta- tion by SH3 binding-deficient NS1 proteins might be rescued by proteins in these lysates was detected with anti-Myc antibody or labeled strepta- CrkL overexpression. vidin. The uniform loading of the lysates was controlled by probing with an anti-α- CrkL overexpression. vidin. The uniform loading of the lysates was controlled by probing with an anti-α- To test this idea we transfected wild-type Mallard NS1 and tubulin antibody (bottom panel). (b) Phospho-Akt signal intensities from (a) and To test this idea we transfected wild-type Mallard NS1 and tubulin antibody (bottom panel). (b) Phospho-Akt signal intensities from (a) and two similar experiments were quantified using the Odyssey scanning software, and two similar experiments were quantified using the Odyssey scanning software, and NS1-AxxA with or without CrkL, and compared the activation of the enhanced pAkt signal intensity is shown as fold increase induced by CrkL NS1-AxxA with or without CrkL, and compared the activation of the enhanced pAkt signal intensity is shown as fold increase induced by CrkL PI3K in these cells (Fig. 6). Overexpression of CrkL had a relatively overexpression as indicated. Statistical significance of the differences were deter- PI3K in these cells (Fig. 6). Overexpression of CrkL had a relatively overexpression as indicated. Statistical significance of the differences were deter- modest enhancing effect on wild-type NS1-induced Akt phosphor- mined by Student's t-test (*Po0.01). modest enhancing effect on wild-type NS1-induced Akt phosphor- mined by Student's t-test (*Po0.01). ylation, whereas the weaker PI3K activation by NS1-AxxA was ylation, whereas the weaker PI3K activation by NS1-AxxA was significantly potentiated by CrkL overexpression. When the natu- significantly potentiated by CrkL overexpression. When the natu- rally Crk(L) binding-deficient NS1 protein from the Udorn strain adapter proteins is associated with enhanced PI3K activation rally Crk(L) binding-deficient NS1 protein from the Udorn strain adapter proteins is associated with enhanced PI3K activation was tested, a similar potentiation of PI3K activation by CrkL (Heikkinen et al., 2008). The same SH3 domain normally couples was tested, a similar potentiation of PI3K activation by CrkL (Heikkinen et al., 2008). The same SH3 domain normally couples overexpression could also be observed. Moreover, we also tested Crk proteins to PI3K by binding to a proline-rich target motif in overexpression could also be observed. Moreover, we also tested Crk proteins to PI3K by binding to a proline-rich target motif in the capacity of overexpressed CrkI and CrkII to potentiate PI3K/Akt p85pre (Gelkop et al., 2001; Sattler et al., 1997). the capacity of overexpressed CrkI and CrkII to potentiate PI3K/Akt p85pre (Gelkop et al., 2001; Sattler et al., 1997). activation by Crk(L) binding-deficient NS1, and obtained results In this study we show that SH3 binding-competent NS1 activation by Crk(L) binding-deficient NS1, and obtained results In this study we show that SH3 binding-competent NS1 that were very similar to those seen with CrkL (data not shown). proteins can displace this natural SH3 interaction, and direct the that were very similar to those seen with CrkL (data not shown). proteins can displace this natural SH3 interaction, and direct the Previously it was reported that the SH3 binding function of NS1 assembly of a novel Crk(L)–NS1–p85β complex (“Complex 1”) Previously it was reported that the SH3 binding function of NS1 assembly of a novel Crk(L)–NS1–p85β complex (“Complex 1”) is involved in regulation of the c-Abl activity, a tyrosine kinase that where NS1 is the bridging factor. The interaction of p85β with is involved in regulation of the c-Abl activity, a tyrosine kinase that where NS1 is the bridging factor. The interaction of p85β with phosphorylates tyrosine residues Y221 and Y207 in Crk and CrkL, NS1 lacking the capacity for SH3 binding also takes place in phosphorylates tyrosine residues Y221 and Y207 in Crk and CrkL, NS1 lacking the capacity for SH3 binding also takes place in respectively (Hrincius et al., 2014). Moreover, c-Abl could also association with Crk(L), but involves an alternative trimeric com- respectively (Hrincius et al., 2014). Moreover, c-Abl could also association with Crk(L), but involves an alternative trimeric com- phosphorylate p85β (Sattler et al., 1996). While an effective plex NS1–p85β–Crk(L) (“Complex 2”) where p85β is the bridging phosphorylate p85β (Sattler et al., 1996). While an effective plex NS1–p85β–Crk(L) (“Complex 2”) where p85β is the bridging inhibition of c-Abl activity was evident by a profound suppression factor. Because of the low cellular levels of p85β, and the relatively inhibition of c-Abl activity was evident by a profound suppression factor. Because of the low cellular levels of p85β, and the relatively of CrkL tyrosine phosphorylation, we observed no effect on NS1- modest affinity of p85β for the CrkL SH3 domain, Complex 2 could of CrkL tyrosine phosphorylation, we observed no effect on NS1- modest affinity of p85β for the CrkL SH3 domain, Complex 2 could induced Akt phosphorylation (Supplementary Fig. 3), thus ruling be physically observed only when p85β was overexpressed. induced Akt phosphorylation (Supplementary Fig. 3), thus ruling be physically observed only when p85β was overexpressed. out a critical role for c-Abl in this regulation. Indeed, our data suggest that the enhanced PI3K activation in out a critical role for c-Abl in this regulation. Indeed, our data suggest that the enhanced PI3K activation in In summary, we conclude that the efficient recruitment of Crk the context of Complex 1 is caused by a more efficient tethering of In summary, we conclude that the efficient recruitment of Crk the context of Complex 1 is caused by a more efficient tethering of proteins into the PI3K–NS1 complex underlies the enhanced PI3K Crk(L) to NS1 and PI3K. In support of this conclusion, overexpres- proteins into the PI3K–NS1 complex underlies the enhanced PI3K Crk(L) to NS1 and PI3K. In support of this conclusion, overexpres- activation by SH3 binding competent NS1 proteins. However, sion of CrkL could compensate for this difference, and thereby activation by SH3 binding competent NS1 proteins. However, sion of CrkL could compensate for this difference, and thereby additional qualitative differences between the two alternative potentiate PI3K activation by SH3 binding-deficient NS1. additional qualitative differences between the two alternative potentiate PI3K activation by SH3 binding-deficient NS1. Crk(L)–NS1–p85β might also exist and could favor PI3K activation These results establish that enhancement of NS1-induced PI3K/ Crk(L)–NS1–p85β might also exist and could favor PI3K activation These results establish that enhancement of NS1-induced PI3K/ in the context of Complex 1. Akt signaling by Crk(L) is directly coupled to the PI3K-activating in the context of Complex 1. Akt signaling by Crk(L) is directly coupled to the PI3K-activating NS1–p85β interaction, rather than acting somewhere upstream or NS1–p85β interaction, rather than acting somewhere upstream or downstream in the PI3K/Akt signaling pathway. Previous studies downstream in the PI3K/Akt signaling pathway. Previous studies Discussion have found both Crk(L) and p85β in anti-NS1 immunocomplexes Discussion have found both Crk(L) and p85β in anti-NS1 immunocomplexes (Heikkinen et al., 2008; Hrincius et al., 2010), but this could also be (Heikkinen et al., 2008; Hrincius et al., 2010), but this could also be Binding to the p85β regulatory subunit of PI3K is a conserved explained by distinct dimeric NS1–p85β and NS1–Crk Binding to the p85β regulatory subunit of PI3K is a conserved explained by distinct dimeric NS1–p85β and NS1–Crk function of NS1 proteins of diverse IAV strains (Ehrhardt and (L) complexes. In contrast, the combination of our mutagenesis function of NS1 proteins of diverse IAV strains (Ehrhardt and (L) complexes. In contrast, the combination of our mutagenesis Ludwig, 2009; Hale and Randall, 2007). This interaction leads to and co-precipitation approaches provides a definite proof for Ludwig, 2009; Hale and Randall, 2007). This interaction leads to and co-precipitation approaches provides a definite proof for activation of PI3K/Akt signaling, which can contribute to viral mutual complexes involving all three proteins, and correlates the activation of PI3K/Akt signaling, which can contribute to viral mutual complexes involving all three proteins, and correlates the replication and disease pathogenesis (see later). The capacity of assembly of this trimeric complex with enhancemement of PI3K/ replication and disease pathogenesis (see later). The capacity of assembly of this trimeric complex with enhancemement of PI3K/ NS1 of certain IAV strains to bind to the SH3 domains of the Crk Akt activation. NS1 of certain IAV strains to bind to the SH3 domains of the Crk Akt activation. L. Ylösmäki et al. / Virology 484 (2015) 146–152 151 L. Ylösmäki et al. / Virology 484 (2015) 146–152 151

Importantly, our results also reveal a general role for Crk of the Crk proteins in order to enhance NS1-mediated PI3K Importantly, our results also reveal a general role for Crk of the Crk proteins in order to enhance NS1-mediated PI3K proteins as host cell cofactors in NS1-mediated activation of activation attests the importance of PI3K-regulated host cell proteins as host cell cofactors in NS1-mediated activation of activation attests the importance of PI3K-regulated host cell PI3K/Akt signaling, which is not limited to NS1 variants carrying processes for IAV. PI3K/Akt signaling, which is not limited to NS1 variants carrying processes for IAV. a functional Crk(L) SH3 binding motif. However, the latter have a functional Crk(L) SH3 binding motif. However, the latter have clearly evolved a more efficient strategy to usurp Crk proteins as clearly evolved a more efficient strategy to usurp Crk proteins as cofactors in PI3K regulation. The mechanistic role of Crk(L) in NS1- cofactors in PI3K regulation. The mechanistic role of Crk(L) in NS1- Materials and methods Materials and methods mediated PI3K/Akt activation remains to be fully understood. It is mediated PI3K/Akt activation remains to be fully understood. It is possible that Crk(L) or an additional Crk(L)-associated protein possible that Crk(L) or an additional Crk(L)-associated protein Cells Cells would be directly involved in the process where binding of NS1 would be directly involved in the process where binding of NS1 leads to dismantling of the negative regulation imposed by p85β leads to dismantling of the negative regulation imposed by p85β Human embryonic kidney 293T (ATCC: CRL-11268) and human Human embryonic kidney 293T (ATCC: CRL-11268) and human on the p110 subunit. Considering the role of Crk(L) as an arche- on the p110 subunit. Considering the role of Crk(L) as an arche- hepatocellular carcinoma Huh-7 (a gift from Mark Harris, Uni- hepatocellular carcinoma Huh-7 (a gift from Mark Harris, Uni- typic cellular adapter protein, as well as the flexibility of the typic cellular adapter protein, as well as the flexibility of the versity of Leeds, UK) cell lines were maintained in Dulbecco's versity of Leeds, UK) cell lines were maintained in Dulbecco's observed Crk(L)–NS1–p85β complex architecture, however, a observed Crk(L)–NS1–p85β complex architecture, however, a modified Eagle's medium high glucose supplemented with 0.6 μg/ modified Eagle's medium high glucose supplemented with 0.6 μg/ perhaps more likely scenario is that Crk(L) serves as an anchor perhaps more likely scenario is that Crk(L) serves as an anchor ml penicillin, 60 μg/ml streptomycin, 10% fetal bovine serum, and ml penicillin, 60 μg/ml streptomycin, 10% fetal bovine serum, and to localize the NS1–PI3K complex to a subcellular compartment to localize the NS1–PI3K complex to a subcellular compartment 2 mM glutamine. 2 mM glutamine. that is favorable for activation of the PI3K/Akt pathway by NS1. that is favorable for activation of the PI3K/Akt pathway by NS1. Indeed, PI3K signaling is complex, involving several alternative Indeed, PI3K signaling is complex, involving several alternative p85 and p110 isoforms that can associate in many different Plasmids and recombinant proteins p85 and p110 isoforms that can associate in many different Plasmids and recombinant proteins combinations (Vanhaesebroeck et al., 2010). It is likely that these combinations (Vanhaesebroeck et al., 2010). It is likely that these different types of PI3K variants operate in overlapping but differ- Plasmid constructs for the myc-NS1 (A/Mallard/Netherlands/ different types of PI3K variants operate in overlapping but differ- Plasmid constructs for the myc-NS1 (A/Mallard/Netherlands/ ent signaling pathways, and associate with distinct sets of partner 12/2000/H7N3, A/Udorn/72 and A/WSN/33) proteins, GST-NS1, ent signaling pathways, and associate with distinct sets of partner 12/2000/H7N3, A/Udorn/72 and A/WSN/33) proteins, GST-NS1, proteins that regulate and target their activities. Thus, it is easy to MBP-NS1 and the C-terminally biotinylated Crk proteins have proteins that regulate and target their activities. Thus, it is easy to MBP-NS1 and the C-terminally biotinylated Crk proteins have – – envision how Crk(L) could be involved in guiding the NS1 PI3K been described before (Heikkinen et al., 2008; Kesti et al., 2007). envision how Crk(L) could be involved in guiding the NS1 PI3K been described before (Heikkinen et al., 2008; Kesti et al., 2007). fi fi complex to a speci c cellular signaling domain that optimally The cDNA for mouse p85β (Open Biosystems) was cloned into the complex to a speci c cellular signaling domain that optimally The cDNA for mouse p85β (Open Biosystems) was cloned into the supports PI3K activation as well as otherwise ensures a cellular pEBB-vector (Tanaka et al., 1995) with an N-terminal HA-tag or a supports PI3K activation as well as otherwise ensures a cellular pEBB-vector (Tanaka et al., 1995) with an N-terminal HA-tag or a fi fi response that maximally bene ts the virus. biotin acceptor domain. Codon changes in NS1 and p85β genes response that maximally bene ts the virus. biotin acceptor domain. Codon changes in NS1 and p85β genes The NS1 gene of most seasonal IAV strains would need just a were generated by standard overlap PCR mutagenesis. GST-NS1 The NS1 gene of most seasonal IAV strains would need just a were generated by standard overlap PCR mutagenesis. GST-NS1 single nucleotide change to acquire the capacity to encode a and MBP-NS1 recombinant proteins were produced as previously single nucleotide change to acquire the capacity to encode a and MBP-NS1 recombinant proteins were produced as previously functional Crk(L) SH3 binding motif, which therefore would be described (Heikkinen et al., 2008). functional Crk(L) SH3 binding motif, which therefore would be described (Heikkinen et al., 2008). expected happen quickly if it provided direct replicative advantage expected happen quickly if it provided direct replicative advantage for the virus. Thus, the reason why viruses like the 1918 pandemic for the virus. Thus, the reason why viruses like the 1918 pandemic strain have incorporated this feature into their genome must Protein precipitation and detection strain have incorporated this feature into their genome must Protein precipitation and detection reflect part of a more complex strategy that they have evolved to reflect part of a more complex strategy that they have evolved to interact with PI3K-dependent pro- and antiviral processes in For immunoprecipitation and protein pull-down experiments, interact with PI3K-dependent pro- and antiviral processes in For immunoprecipitation and protein pull-down experiments, their hosts. 293T cells were transfected by a standard calcium phosphate their hosts. 293T cells were transfected by a standard calcium phosphate Regulation of host cell PI3K activity during different steps of precipitation method. 48 h after transfection the cells were col- Regulation of host cell PI3K activity during different steps of precipitation method. 48 h after transfection the cells were col- the IAV life-cycle is a multifaceted process that is not limited to the lected and lysed in 1% NP40 lysis buffer (150 mM NaCl; 50 mM the IAV life-cycle is a multifaceted process that is not limited to the lected and lysed in 1% NP40 lysis buffer (150 mM NaCl; 50 mM action of NS1 (see Ehrhardt and Ludwig, 2009). The role of PI3K Tris–HCl, pH 7.9; 1% NP40). Cell lysates were used for immuno- action of NS1 (see Ehrhardt and Ludwig, 2009). The role of PI3K Tris–HCl, pH 7.9; 1% NP40). Cell lysates were used for immuno- activation by NS1 during IAV replication in cell culture and precipitation with an anti-CrkL or an anti-HA antibody coupled to activation by NS1 during IAV replication in cell culture and precipitation with an anti-CrkL or an anti-HA antibody coupled to pathogenesis in mice has been studied using engineered viruses Dynabeads protein G magnetic beads (Invitrogen). Alternately pathogenesis in mice has been studied using engineered viruses Dynabeads protein G magnetic beads (Invitrogen). Alternately with mutant NS1 proteins that cannot bind to p85β. Originally it lysates were used for streptavidin pulldown with streptavidin- with mutant NS1 proteins that cannot bind to p85β. Originally it lysates were used for streptavidin pulldown with streptavidin- was reported that such mutants are attenuated, giving rise to a coated Dynabeads (Invitrogen). To examine the phosphorylation was reported that such mutants are attenuated, giving rise to a coated Dynabeads (Invitrogen). To examine the phosphorylation smaller plaque size and lower viral titers (Hale et al., 2006; Shin et status of Akt, Huh7 cells in 6-well plates were transfected with smaller plaque size and lower viral titers (Hale et al., 2006; Shin et status of Akt, Huh7 cells in 6-well plates were transfected with al., 2007). More recently a profound reduction in viral replication 4 mg of plasmid DNA using Lipofectamine 2000 (Invitrogen) al., 2007). More recently a profound reduction in viral replication 4 mg of plasmid DNA using Lipofectamine 2000 (Invitrogen) and pathogenesis in mouse models of IAV infection was also according to manufacturer's instructions. Cells were serum- and pathogenesis in mouse models of IAV infection was also according to manufacturer's instructions. Cells were serum- reported (Ayllon et al., 2012b; Hrincius et al., 2012). However, starved for 12 h and 48 h after transfection followed by lysis in reported (Ayllon et al., 2012b; Hrincius et al., 2012). However, starved for 12 h and 48 h after transfection followed by lysis in the study by Hale and colleagues showed that the requirement for 1% NP40 buffer. In Supplementary Fig. 3 the cells were treated the study by Hale and colleagues showed that the requirement for 1% NP40 buffer. In Supplementary Fig. 3 the cells were treated NS1-induced PI3K activation is strikingly virus strain dependent. with 20 μM Imatinib for 12 h. Western blots were visualized with NS1-induced PI3K activation is strikingly virus strain dependent. with 20 μM Imatinib for 12 h. Western blots were visualized with While the underlying biology remains to be clarified (see Ayllon et the Odyssey infrared imaging system (LI-COR Biosciences). While the underlying biology remains to be clarified (see Ayllon et the Odyssey infrared imaging system (LI-COR Biosciences). al., 2012a), it is evident that variation elsewhere in the viral al., 2012a), it is evident that variation elsewhere in the viral genome can render PI3K activation by NS1 largely or completely genome can render PI3K activation by NS1 largely or completely dispensable for IAV replication. On the other hand, only some Antibodies and inhibitors dispensable for IAV replication. On the other hand, only some Antibodies and inhibitors (typically avian) strains of IAV encode SH3-binding competent NS1 (typically avian) strains of IAV encode SH3-binding competent NS1 proteins that can efficiently recruit Crk proteins to enhance NS1- The following primary antibodies were used in this study: proteins that can efficiently recruit Crk proteins to enhance NS1- The following primary antibodies were used in this study: induced PI3K activation (Heikkinen et al., 2008). As shown in the mouse anti-Myc (9E10, Santa Cruz Biotechnology), mouse anti- induced PI3K activation (Heikkinen et al., 2008). As shown in the mouse anti-Myc (9E10, Santa Cruz Biotechnology), mouse anti- current study, however, Crk(L)-mediated potentiation of PI3K/Akt CrkL (clone 5–6, Millipore), mouse anti-HA (F-7, Santa Cruz current study, however, Crk(L)-mediated potentiation of PI3K/Akt CrkL (clone 5–6, Millipore), mouse anti-HA (F-7, Santa Cruz signaling depends entirely on p85β-binding coordinated by the Biotechnology), rabbit anti-phospho Akt(Ser473) (D9E, Cell Signal- signaling depends entirely on p85β-binding coordinated by the Biotechnology), rabbit anti-phospho Akt(Ser473) (D9E, Cell Signal- tyrosine residue 89 of NS1. Thus, the variable capacity for Crk ing Technology), mouse anti-Akt (40D4, Cell Signaling Technol- tyrosine residue 89 of NS1. Thus, the variable capacity for Crk ing Technology), mouse anti-Akt (40D4, Cell Signaling Technol- (L) SH3 binding by NS1 proteins from different IAV provides ogy), mouse anti-p85β (T15, AbD Serotec), mouse anti-α-tubulin (L) SH3 binding by NS1 proteins from different IAV provides ogy), mouse anti-p85β (T15, AbD Serotec), mouse anti-α-tubulin another layer of complexity to the strain-specific variation in host (DM1A, Sigma-Aldrich) and guinea-pig anti-NS1 (Melen et al., another layer of complexity to the strain-specific variation in host (DM1A, Sigma-Aldrich) and guinea-pig anti-NS1 (Melen et al., cell PI3K regulation. 2007). Streptavidin IRDye680CW, Streptavidin IRDye800CW, cell PI3K regulation. 2007). Streptavidin IRDye680CW, Streptavidin IRDye800CW, In summary, the data presented in this study provide a detailed IRDye680CW goat anti-mouse IgG, IRDye800CW goat anti-mouse In summary, the data presented in this study provide a detailed IRDye680CW goat anti-mouse IgG, IRDye800CW goat anti-mouse characterization of the Crk(L)–NS1–PI3K multiprotein complex, IgG, IRDye680CW goat anti-rabbit IgG, and IRDye800CW goat anti- characterization of the Crk(L)–NS1–PI3K multiprotein complex, IgG, IRDye680CW goat anti-rabbit IgG, and IRDye800CW goat anti- and emphasize the role of Crk proteins as host cell cofactors of the rabbit were from LI-COR Biotechnology. IRDye800CW rabbit anti- and emphasize the role of Crk proteins as host cell cofactors of the rabbit were from LI-COR Biotechnology. IRDye800CW rabbit anti- IAV virulence factor NS1. The fact that some IAV have incorporated guinea pig was from Rockland Immunochemicals. The c-Abl IAV virulence factor NS1. The fact that some IAV have incorporated guinea pig was from Rockland Immunochemicals. The c-Abl a Crk(L) SH3 binding motif in the NS1 protein to take maximal use inhibitor Imatinib was from Sigma-Aldrich. a Crk(L) SH3 binding motif in the NS1 protein to take maximal use inhibitor Imatinib was from Sigma-Aldrich. 152 L. Ylösmäki et al. / Virology 484 (2015) 146–152 152 L. Ylösmäki et al. / Virology 484 (2015) 146–152

Acknowledgments Heikkinen, L.S., Kazlauskas, A., Melen, K., Wagner, R., Ziegler, T., Julkunen, I., Acknowledgments Heikkinen, L.S., Kazlauskas, A., Melen, K., Wagner, R., Ziegler, T., Julkunen, I., Saksela, K., 2008. Avian and 1918 Spanish influenza a virus NS1 proteins bind Saksela, K., 2008. Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling. J. Biol. to Crk/CrkL Src homology 3 domains to activate host cell signaling. J. Biol. This study was supported by Grants to K.S. from the Academy Chem. 283, 5719–5727 (Epub 2007 Dec 5728). This study was supported by Grants to K.S. from the Academy Chem. 283, 5719–5727 (Epub 2007 Dec 5728). of Finland, Helsinki University Central Hospital Research Council, Hrincius, E.R., Dierkes, R., Anhlan, D., Wixler, V., Ludwig, S., Ehrhardt, C., 2011. of Finland, Helsinki University Central Hospital Research Council, Hrincius, E.R., Dierkes, R., Anhlan, D., Wixler, V., Ludwig, S., Ehrhardt, C., 2011. Biocentrum Helsinki, and the Sigrid Juselius Foundation. L.Y. and C. Phosphatidylinositol-3-kinase (PI3K) is activated by influenza virus vRNA via Biocentrum Helsinki, and the Sigrid Juselius Foundation. L.Y. and C. Phosphatidylinositol-3-kinase (PI3K) is activated by influenza virus vRNA via fi fi S. were supported in part by University of Helsinki Doctoral School the pathogen pattern receptor Rig-I to promote ef cient type I interferon S. were supported in part by University of Helsinki Doctoral School the pathogen pattern receptor Rig-I to promote ef cient type I interferon production. Cell. Microbiol.. production. Cell. Microbiol.. in Health Sciences. We thank Virpi Syvälahti for technical Hrincius, E.R., Hennecke, A.K., Gensler, L., Nordhoff, C., Anhlan, D., Vogel, P., in Health Sciences. We thank Virpi Syvälahti for technical Hrincius, E.R., Hennecke, A.K., Gensler, L., Nordhoff, C., Anhlan, D., Vogel, P., assistance. McCullers, J.A., Ludwig, S., Ehrhardt, C., 2012. A single point mutation (Y89F) assistance. McCullers, J.A., Ludwig, S., Ehrhardt, C., 2012. A single point mutation (Y89F) within the non-structural protein 1 of influenza A viruses limits epithelial cell within the non-structural protein 1 of influenza A viruses limits epithelial cell tropism and virulence in mice. Am. J. Pathol. 180, 2361–2374. tropism and virulence in mice. Am. J. Pathol. 180, 2361–2374. Appendix A. Supporting information Hrincius, E.R., Liedmann, S., Anhlan, D., Wolff, T., Ludwig, S., Ehrhardt, C., 2014. Appendix A. Supporting information Hrincius, E.R., Liedmann, S., Anhlan, D., Wolff, T., Ludwig, S., Ehrhardt, C., 2014. Avian influenza viruses inhibit the major cellular signalling integrator c-Abl. Avian influenza viruses inhibit the major cellular signalling integrator c-Abl. Cell. Microbiol. 16, 1854–1874. Cell. Microbiol. 16, 1854–1874. Supplementary data associated with this article can be found in Hrincius, E.R., Wixler, V., Wolff, T., Wagner, R., Ludwig, S., Ehrhardt, C., 2010. 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Supplementary Fig. 1. Supplementary Fig. 1. Competitive disruption of the CrkL/p85β-V573M complex by titration of recombinant wild- Competitive disruption of the CrkL/p85β-V573M complex by titration of recombinant wild- type NS1. Increasing amounts (from 0 μg to 12 μg) of recombinant wild-type NS1 or 12 μg of NS1- type NS1. Increasing amounts (from 0 μg to 12 μg) of recombinant wild-type NS1 or 12 μg of NS1- AxxA expressed as GST fusion proteins were added to lysates of cells transfected with biotinylation AxxA expressed as GST fusion proteins were added to lysates of cells transfected with biotinylation domain-tagged CrkL and HA-tagged p85β-V573M (total protein amount 200 μg). The amount of domain-tagged CrkL and HA-tagged p85β-V573M (total protein amount 200 μg). The amount of p85β and NS1 proteins associated with CrkL precipitated with streptavidin beads was examined by p85β and NS1 proteins associated with CrkL precipitated with streptavidin beads was examined by Western blotting. Western blotting.

Supplementary Fig. 2. Supplementary Fig. 2. Complex formation with purified proteins. (a) Biotinylation domain-tagged wild-type and Complex formation with purified proteins. (a) Biotinylation domain-tagged wild-type and P294A/P297A mutant p85β proteins (200 ng per reaction) purified from 293 cell cultures were P294A/P297A mutant p85β proteins (200 ng per reaction) purified from 293 cell cultures were mixed as indicated with MBP-tagged wild-type or AxxA mutant NS1 proteins (500 ng per reaction) mixed as indicated with MBP-tagged wild-type or AxxA mutant NS1 proteins (500 ng per reaction) and/or GST-tagged SH3 domain-containg fragment of CrkL (2 μg per reaction). NS1 and CrkL and/or GST-tagged SH3 domain-containg fragment of CrkL (2 μg per reaction). NS1 and CrkL proteins co-precipitating with p85β proteins on streptavidin-coated particles were detected with an proteins co-precipitating with p85β proteins on streptavidin-coated particles were detected with an anti-NS1 antibody (middle panel) and anti-GST antibody (bottom panel). Despite some unavoidable anti-NS1 antibody (middle panel) and anti-GST antibody (bottom panel). Despite some unavoidable background binding (evident also in the absence of any NS1) of the GST-tagged CrkL fragment to background binding (evident also in the absence of any NS1) of the GST-tagged CrkL fragment to the p85β-P294A/P297A-containing beads, the specific capacity of wild-type NS1 (but not NS1- the p85β-P294A/P297A-containing beads, the specific capacity of wild-type NS1 (but not NS1- AxxA) to couple CrkL to p85β-P294A/P297A is clearly evident. (b) Quality and purity of the AxxA) to couple CrkL to p85β-P294A/P297A is clearly evident. (b) Quality and purity of the proteins used in (a) analyzed by SDS-PAGE and Coomassie staining. proteins used in (a) analyzed by SDS-PAGE and Coomassie staining.

Supplementary Fig. 3. Supplementary Fig. 3. Effect of c-Abl inhibition on PI3K/Akt activation induced by NS1. Wild-type Mallard NS1 Effect of c-Abl inhibition on PI3K/Akt activation induced by NS1. Wild-type Mallard NS1 (WT) or an empty control vector (vector) was transfected into Huh7 cells. The cells were serum (WT) or an empty control vector (vector) was transfected into Huh7 cells. The cells were serum starved and treated or left untreated with 20 μM Imatinib for 12 h. Induction of PI3K/Akt signaling starved and treated or left untreated with 20 μM Imatinib for 12 h. Induction of PI3K/Akt signaling was examined by probing the lysates with an anti-phospho-Akt antibody (top panel), and expression was examined by probing the lysates with an anti-phospho-Akt antibody (top panel), and expression of NS1 detected with an anti-Myc antibody (upper middle panel). To control the effect of Imatinib of NS1 detected with an anti-Myc antibody (upper middle panel). To control the effect of Imatinib treatment, antibody specific for phospho-CrkL (Tyr207) was used (lower middle panel). Even treatment, antibody specific for phospho-CrkL (Tyr207) was used (lower middle panel). Even loading of the gel was confirmed with an antibody for cellular α-tubulin (bottom panel). loading of the gel was confirmed with an antibody for cellular α-tubulin (bottom panel). III III

viruses viruses

Article Article Nuclear Translocation of Crk Adaptor Proteins by the Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein Influenza A Virus NS1 Protein

Leena Ylösmäki 1, Riku Fagerlund 1, Inka Kuisma 1, Ilkka Julkunen 2 and Kalle Saksela 1,* Leena Ylösmäki 1, Riku Fagerlund 1, Inka Kuisma 1, Ilkka Julkunen 2 and Kalle Saksela 1,*

1 Department of Virology, University of Helsinki and Helsinki University Hospital, 00014 Helsinki, Finland; 1 Department of Virology, University of Helsinki and Helsinki University Hospital, 00014 Helsinki, Finland; leena.ylosmaki@helsinki.fi (L.Y.); riku.fagerlund@helsinki.fi (R.F.); inka.kuisma@helsinki.fi (I.K.) leena.ylosmaki@helsinki.fi (L.Y.); riku.fagerlund@helsinki.fi (R.F.); inka.kuisma@helsinki.fi (I.K.) 2 Department of Virology, University of Turku, 20520 Turku, Finland and Virology Unit, Department of 2 Department of Virology, University of Turku, 20520 Turku, Finland and Virology Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare (THL), 00300 Helsinki, Infectious Disease Surveillance and Control, National Institute for Health and Welfare (THL), 00300 Helsinki, Finland; ilkka.julkunen@utu.fi Finland; ilkka.julkunen@utu.fi * Correspondence: kalle.saksela@helsinki.fi; Tel.: +358-2-9412-6770 * Correspondence: kalle.saksela@helsinki.fi; Tel.: +358-2-9412-6770

Academic Editor: Andrew Mehle Academic Editor: Andrew Mehle Received: 9 February 2016; Accepted: 4 April 2016; Published: 15 April 2016 Received: 9 February 2016; Accepted: 4 April 2016; Published: 15 April 2016

Abstract: The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian Abstract: The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif. cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif.

Keywords: NS1; influenza A virus; SH3 domain; Crk; virus-host interaction Keywords: NS1; influenza A virus; SH3 domain; Crk; virus-host interaction

1. Introduction 1. Introduction

Influenza A virus (IAV) belongs to the Orthomyxoviridae family of enveloped viruses. It has a Influenza A virus (IAV) belongs to the Orthomyxoviridae family of enveloped viruses. It has a segmented genome consisting of eight single stranded negative-sense RNA strands. The non-structural segmented genome consisting of eight single stranded negative-sense RNA strands. The non-structural protein 1 (NS1) of IAV is an important virulence factor, and a remarkably multifunctional protein that protein 1 (NS1) of IAV is an important virulence factor, and a remarkably multifunctional protein that acts in several different ways to facilitate IAV replication (for reviews, see [1,2]). acts in several different ways to facilitate IAV replication (for reviews, see [1,2]). The dynamic localization of NS1 in the nucleus as well as in the cytoplasm of IAV-infected cells The dynamic localization of NS1 in the nucleus as well as in the cytoplasm of IAV-infected cells is mediated by two nuclear localization signals (NLS) and by one nuclear export signal (NES) [3–5]. is mediated by two nuclear localization signals (NLS) and by one nuclear export signal (NES) [3–5]. Soon after IAV infection, newly synthesized NS1 accumulates in the nucleus, but at late time points Soon after IAV infection, newly synthesized NS1 accumulates in the nucleus, but at late time points of infection it is transported into the cytoplasm. The conserved NLS1 of NS1 protein involves the of infection it is transported into the cytoplasm. The conserved NLS1 of NS1 protein involves the amino acids R35, R37, R38, and K41 [3,6], while NLS2 is virus strain-specific, and it is located in the amino acids R35, R37, R38, and K41 [3,6], while NLS2 is virus strain-specific, and it is located in the C-terminus of the protein [3,6,7]. The NES is located between the amino acids 138–147, leucine residues C-terminus of the protein [3,6,7]. The NES is located between the amino acids 138–147, leucine residues 144 and 146 being critical for its function [8,9]. 144 and 146 being critical for its function [8,9]. The NS1 protein has several reported functions both in the nucleus and in the cytoplasm. The NS1 protein has several reported functions both in the nucleus and in the cytoplasm. In the nucleus, NS1 can inhibit cellular mRNA maturation and export by interacting with cleavage In the nucleus, NS1 can inhibit cellular mRNA maturation and export by interacting with cleavage and polyadenylation specificity factor (CPSF), poly(A)-binding protein II (PABPII), mRNA splicing and polyadenylation specificity factor (CPSF), poly(A)-binding protein II (PABPII), mRNA splicing machinery, and nuclear export factors [10–12]. In the cytoplasm, NS1 prevents the activation of machinery, and nuclear export factors [10–12]. In the cytoplasm, NS1 prevents the activation of interferon-inducing proteins by inhibiting RNA helicase retinoic acid inducible gene-I (RIG-I) through interferon-inducing proteins by inhibiting RNA helicase retinoic acid inducible gene-I (RIG-I) through a direct interaction [13,14], and by preventing RIG-I ubiquitination via interacting with ubiquitin E3 a direct interaction [13,14], and by preventing RIG-I ubiquitination via interacting with ubiquitin E3

Viruses 2016, 8, 101; doi:10.3390/v8040101 www.mdpi.com/journal/viruses Viruses 2016, 8, 101; doi:10.3390/v8040101 www.mdpi.com/journal/viruses Viruses 2016, 8, 101 2 of 15 Viruses 2016, 8, 101 2 of 15 ligases TRIM-25 and Riplet, [15,16]. NS1 also inhibits the activity of protein kinase R (PKR) [17], and ligases TRIM-25 and Riplet, [15,16]. NS1 also inhibits the activity of protein kinase R (PKR) [17], and 21-51-oligoadenylate synthetase (OAS) [18], two important interferon-induced antiviral proteins. 21-51-oligoadenylate synthetase (OAS) [18], two important interferon-induced antiviral proteins. In addition, NS1 can activate the host cell phosphatidylinositol 3-kinase (PI3K) cascade, a signaling In addition, NS1 can activate the host cell phosphatidylinositol 3-kinase (PI3K) cascade, a signaling pathway intimately involved in viral replication and innate immunity, by interacting directly with pathway intimately involved in viral replication and innate immunity, by interacting directly with p85β, a regulatory subunit of the PI3K complex [19,20]. PI3K activation is further enhanced by NS1 p85β, a regulatory subunit of the PI3K complex [19,20]. PI3K activation is further enhanced by NS1 proteins that contain an SH3 binding motif, which mediates a strong and selective binding to the proteins that contain an SH3 binding motif, which mediates a strong and selective binding to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adaptor protein (CrkL) [21]. This NS1 SH3 binding motif is commonly found in avian IAVs, but adaptor protein (CrkL) [21]. This NS1 SH3 binding motif is commonly found in avian IAVs, but only in some human IAV strains, including the 1918 pandemic Spanish flu virus. This potentiation only in some human IAV strains, including the 1918 pandemic Spanish flu virus. This potentiation of PI3K activation involves reorganization of the cellular p85β-Crk protein complex. While SH3 of PI3K activation involves reorganization of the cellular p85β-Crk protein complex. While SH3 binding-incompetent NS1 proteins simply bind to p85β in this complex, PI3K-superactivating NS1 binding-incompetent NS1 proteins simply bind to p85β in this complex, PI3K-superactivating NS1 proteins hijack the SH3 domain of Crk, thereby breaking the pre-existing p85β-Crk complex and proteins hijack the SH3 domain of Crk, thereby breaking the pre-existing p85β-Crk complex and assembling an alternative trimeric complex where NS1 is a bridging factor between p85β and Crk [22]. assembling an alternative trimeric complex where NS1 is a bridging factor between p85β and Crk [22]. Crk proteins consist of a family of three members: CrkI, CrkII, and CrkL. CrkII and CrkL both Crk proteins consist of a family of three members: CrkI, CrkII, and CrkL. CrkII and CrkL both contain one SH2 and two SH3 domains, while CrkI is a truncated form of CrkII that due to an alternative contain one SH2 and two SH3 domains, while CrkI is a truncated form of CrkII that due to an alternative mRNA splicing possess only the SH2 and the N-terminal SH3 domain [23,24]. Although Crk proteins mRNA splicing possess only the SH2 and the N-terminal SH3 domain [23,24]. Although Crk proteins lack any enzymatic activity, they play a crucial role in cell biology by serving as essential adaptor lack any enzymatic activity, they play a crucial role in cell biology by serving as essential adaptor proteins linking together different signaling molecules, such as tyrosine kinases and small G proteins proteins linking together different signaling molecules, such as tyrosine kinases and small G proteins through their SH2 and SH3 domains. They coordinate numerous biological processes, ranging from through their SH2 and SH3 domains. They coordinate numerous biological processes, ranging from cell proliferation, cell adhesion and migration, phagocytic and endocytic pathways, apoptosis, and cell proliferation, cell adhesion and migration, phagocytic and endocytic pathways, apoptosis, and regulation of gene expression (for reviews, see [25,26]). The SH2 and SH3 domains of Crk proteins regulation of gene expression (for reviews, see [25,26]). The SH2 and SH3 domains of Crk proteins are highly homologous and display similar binding preferences and they have several overlapping are highly homologous and display similar binding preferences and they have several overlapping roles, for example, in maintaining the cell structure and motility in mouse embryonic fibroblast (MEF) roles, for example, in maintaining the cell structure and motility in mouse embryonic fibroblast (MEF) cells [27]. Use of knockout mice has revealed also some non-overlapping roles for these proteins in cells [27]. Use of knockout mice has revealed also some non-overlapping roles for these proteins in embryonic development. Knockout of CrkI/II or CrkL individually leads to different developmental embryonic development. Knockout of CrkI/II or CrkL individually leads to different developmental defects in mice and they die perinatally [28,29]. Most of the cellular functions described for Crk defects in mice and they die perinatally [28,29]. Most of the cellular functions described for Crk proteins involve coordination of cytoplasmic signaling processes. However, Crk proteins have also proteins involve coordination of cytoplasmic signaling processes. However, Crk proteins have also been reported to enter the nucleus to regulate additional signaling pathways involved in malignant been reported to enter the nucleus to regulate additional signaling pathways involved in malignant transformation and programmed cell death. The nuclear partners for Crk proteins are not well transformation and programmed cell death. The nuclear partners for Crk proteins are not well known, but prominently include the tyrosine kinase c-Abl, whose nuclear functions are important known, but prominently include the tyrosine kinase c-Abl, whose nuclear functions are important in cellular responses to DNA damage, cell cycle progression, and apoptosis [30]. Moreover, nuclear in cellular responses to DNA damage, cell cycle progression, and apoptosis [30]. Moreover, nuclear translocation of CrkII and its interaction with the nuclear tyrosine kinase Wee1 has been reported to translocation of CrkII and its interaction with the nuclear tyrosine kinase Wee1 has been reported to be proapoptotic [31,32]. It has also been reported that the binding of CrkL to phosphorylated form of be proapoptotic [31,32]. It has also been reported that the binding of CrkL to phosphorylated form of signal transducer and activator of transcription (STAT5) leads to translocation of the complex into the signal transducer and activator of transcription (STAT5) leads to translocation of the complex into the nucleus where it binds to the promoter region of c-Abl or Bcr-Abl genes in chronic myeloid leukemia nucleus where it binds to the promoter region of c-Abl or Bcr-Abl genes in chronic myeloid leukemia (CML) cells [33,34]. Regulation of the nuclear entry of Crk proteins is not well understood. CrkII and (CML) cells [33,34]. Regulation of the nuclear entry of Crk proteins is not well understood. CrkII and CrkL have a nuclear export signal located in theC-terminal SH3-domain [35], but all Crk proteins CrkL have a nuclear export signal located in theC-terminal SH3-domain [35], but all Crk proteins lack a canonical nuclear localization signal, and apparently they can enter the nucleus only through lack a canonical nuclear localization signal, and apparently they can enter the nucleus only through interaction with other proteins that contain a functional NLS [36]. interaction with other proteins that contain a functional NLS [36]. Since both NS1 and Crk have distinct nuclear and cytoplasmic functions, and since the effects Since both NS1 and Crk have distinct nuclear and cytoplasmic functions, and since the effects on cellular physiology described for nuclear Crk proteins appear to depend on interaction partners on cellular physiology described for nuclear Crk proteins appear to depend on interaction partners that are actively transported into the nucleus, we examined how NS1 might influence the intracellular that are actively transported into the nucleus, we examined how NS1 might influence the intracellular distribution of Crk proteins. Here we report that infection of cells with IAV encoding NS1 proteins distribution of Crk proteins. Here we report that infection of cells with IAV encoding NS1 proteins that are competent for Crk binding, in contrast to viruses encoding NS1 lacking the SH3 ligand motif, that are competent for Crk binding, in contrast to viruses encoding NS1 lacking the SH3 ligand motif, cause a robust translocation of Crk proteins from the cytoplasm into the nucleus, which is associated cause a robust translocation of Crk proteins from the cytoplasm into the nucleus, which is associated with a noticeable change in tyrosine phosphorylation pattern of proteins in the nuclear fraction. with a noticeable change in tyrosine phosphorylation pattern of proteins in the nuclear fraction. Viruses 2016, 8, 101 3 of 15 Viruses 2016, 8, 101 3 of 15

2. Materials and Methods 2. Materials and Methods

2.1. Cell Culture 2.1. Cell Culture The human lung epithelial (A549) and the human hepatocellular carcinoma (Huh-7) cell lines The human lung epithelial (A549) and the human hepatocellular carcinoma (Huh-7) cell lines were maintained in Dulbecco1s Modified Eagle Medium (DMEM) (Sigma Aldrich, St. Louis, MO, USA) were maintained in Dulbecco1s Modified Eagle Medium (DMEM) (Sigma Aldrich, St. Louis, MO, USA) supplemented with 4500 mg/L of glucose, 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), supplemented with 4500 mg/L of glucose, 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), 0.05 mg/mL penicillin, 0.05 mg/mL streptomycin (Sigma Aldrich), and 1 mM L-glutamine (Sigma 0.05 mg/mL penicillin, 0.05 mg/mL streptomycin (Sigma Aldrich), and 1 mM L-glutamine (Sigma ˝ ˝ Aldrich) at 37 C in 5% CO2. Aldrich) at 37 C in 5% CO2.

2.2. Recombinant Influenza A Viruses 2.2. Recombinant Influenza A Viruses The recombinant influenza A viruses were generated by using a plasmid-based reverse genetics The recombinant influenza A viruses were generated by using a plasmid-based reverse genetics as previously described [37]. A/WSN/1933 IAV was used as the background virus. The NS as previously described [37]. A/WSN/1933 IAV was used as the background virus. The NS segment originated from either A/WSN/1933/H1N1 or A/Mallard/Netherlands/12/2000/H7N3 segment originated from either A/WSN/1933/H1N1 or A/Mallard/Netherlands/12/2000/H7N3 virus. The codon changes to NS1 sequence (A/WSN T215P; A/Mallard K217E) were introduced using virus. The codon changes to NS1 sequence (A/WSN T215P; A/Mallard K217E) were introduced using overlapping polymerase chain reaction (PCR) mutagenesis. Influenza A/WSN/1933 recombinant overlapping polymerase chain reaction (PCR) mutagenesis. Influenza A/WSN/1933 recombinant viruses were propagated in 11-day-old embryonated chicken eggs at 34 ˝C for three days. viruses were propagated in 11-day-old embryonated chicken eggs at 34 ˝C for three days. The recombinant viruses used in this study are: A/WSN-NS1Mallard(wt), A/WSN-NS1Mallard(K217E), The recombinant viruses used in this study are: A/WSN-NS1Mallard(wt), A/WSN-NS1Mallard(K217E), A/WSN-NS1WSN(wt), and A/WSN-NS1WSN(T215P). A/WSN-NS1WSN(wt), and A/WSN-NS1WSN(T215P).

2.3. DNA Transfections and Plasmids 2.3. DNA Transfections and Plasmids A549 and Huh-7 cells were transfected by using a Lipofectamine 2000 reagent (Invitrogen, A549 and Huh-7 cells were transfected by using a Lipofectamine 2000 reagent (Invitrogen, Waltham, Massachusetts, USA) according to manufacturer1s instructions. The vector for A/Mallard Waltham, Massachusetts, USA) according to manufacturer1s instructions. The vector for A/Mallard myc-NS1 wild-type (WT) has been described before [21]. To generate fluorescent fusion proteins, myc-NS1 wild-type (WT) has been described before [21]. To generate fluorescent fusion proteins, mCherry was fused to the N-terminus of A/Mallard NS1, and enhanced green fluorescent protein mCherry was fused to the N-terminus of A/Mallard NS1, and enhanced green fluorescent protein (eGFP) to the N-terminus of CrkL. To generate a cytoplasmic A/Mallard NS1 (Cyto), the NES from (eGFP) to the N-terminus of CrkL. To generate a cytoplasmic A/Mallard NS1 (Cyto), the NES from MAPKK1 (LQKKLEELEL) was inserted between the mCherry and NS1 coding sequences. In addition, MAPKK1 (LQKKLEELEL) was inserted between the mCherry and NS1 coding sequences. In addition, the NLS1 of NS1 protein was mutated (R38A, R41A) by standard overlap PCR mutagenesis. All plasmid the NLS1 of NS1 protein was mutated (R38A, R41A) by standard overlap PCR mutagenesis. All plasmid constructs were verified correct by DNA sequencing. constructs were verified correct by DNA sequencing.

2.4. Antibodies 2.4. Antibodies The following primary antibodies were used in this study: mouse monoclonal anti-CrkL (clone 5–6, The following primary antibodies were used in this study: mouse monoclonal anti-CrkL (clone 5–6, Millipore, Billerica, MA, USA), mouse monoclonal anti-Crk (clone 22, BD Transduction Laboratories, Millipore, Billerica, MA, USA), mouse monoclonal anti-Crk (clone 22, BD Transduction Laboratories, San Jose, CA, USA), rabbit monoclonal anti-phospho Akt (Ser473) (D9E, Cell Signaling Technology, San Jose, CA, USA), rabbit monoclonal anti-phospho Akt (Ser473) (D9E, Cell Signaling Technology, Danver, MA, USA), mouse monoclonalanti-α-tubulin (DM1A, Sigma-Aldrich), rabbit polyclonal Danver, MA, USA), mouse monoclonalanti-α-tubulin (DM1A, Sigma-Aldrich), rabbit polyclonal anti-Histone H3 (Cell Signaling Technology), monoclonal mouse anti-phosphotyrosine (PY20, Santa anti-Histone H3 (Cell Signaling Technology), monoclonal mouse anti-phosphotyrosine (PY20, Santa Cruz Biotechnology, Dallas, TX, USA), and guinea-pig polyclonal anti-NS1 [3]. The secondary Cruz Biotechnology, Dallas, TX, USA), and guinea-pig polyclonal anti-NS1 [3]. The secondary antibodies for Western blotting were: IRDye680CW goat anti-mouse IgG, IRDye680CW goat anti-rabbit antibodies for Western blotting were: IRDye680CW goat anti-mouse IgG, IRDye680CW goat anti-rabbit IgG, and IRDye800CW goat anti-rabbit, and IRDye800CW rabbit anti-guinea pig were from LI-COR IgG, and IRDye800CW goat anti-rabbit, and IRDye800CW rabbit anti-guinea pig were from LI-COR Biotechnology (Lincoln, NE, USA). Secondary antibodies for immunofluorescence staining were: Biotechnology (Lincoln, NE, USA). Secondary antibodies for immunofluorescence staining were: AlexaFluor 488 goat anti-guinea pig IgG (Abcam, Cambridge, UK), and AlexaFluor 546 goat anti-mouse AlexaFluor 488 goat anti-guinea pig IgG (Abcam, Cambridge, UK), and AlexaFluor 546 goat anti-mouse IgG (Molecular Probes, Eugene, OR, USA). Nuclei were stained with Hoechst. IgG (Molecular Probes, Eugene, OR, USA). Nuclei were stained with Hoechst.

2.5. Immunoprecipitation and Detection 2.5. Immunoprecipitation and Detection For immunoprecipitation A549 cells were infected with recombinant IAVs for 24 h, and the cells For immunoprecipitation A549 cells were infected with recombinant IAVs for 24 h, and the cells were collected and lysed in 1% NP40 lysis buffer (150 mM NaCl; 50 mM Tris–HCl, pH 7.9; 1% NP40). were collected and lysed in 1% NP40 lysis buffer (150 mM NaCl; 50 mM Tris–HCl, pH 7.9; 1% NP40). Cell lysates were used for immunoprecipitation with an anti-CrkL antibody coupled to Dynabeads Cell lysates were used for immunoprecipitation with an anti-CrkL antibody coupled to Dynabeads protein G magnetic beads (Invitrogen). To examine the phosphorylation status of Akt, Huh7 cells protein G magnetic beads (Invitrogen). To examine the phosphorylation status of Akt, Huh7 cells on 6-well plates were transfected with 4 µg of plasmid DNA. Transfected cells were serum-starved on 6-well plates were transfected with 4 µg of plasmid DNA. Transfected cells were serum-starved Viruses 2016, 8, 101 4 of 15 Viruses 2016, 8, 101 4 of 15 for 12 h, and 48 h after transfection the cells were lysed in 1% NP40 lysis buffer. Western blots were for 12 h, and 48 h after transfection the cells were lysed in 1% NP40 lysis buffer. Western blots were visualized with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). visualized with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

2.6. Cell Fractionation 2.6. Cell Fractionation A549 cells were seeded on 10 cm diameter well plates at 3 ˆ 106 density. The next day, the cells A549 cells were seeded on 10 cm diameter well plates at 3 ˆ 106 density. The next day, the cells were mock infected or infected with recombinant IAVs at a multiplicity of infections (MOI) 2 in the were mock infected or infected with recombinant IAVs at a multiplicity of infections (MOI) 2 in the presence of 5 µg/mL of N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)-treated trypsin presence of 5 µg/mL of N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma Aldrich). 24 h after infection the cells were scraped into 500 µL of ice cold Buffer A (20 mM (Sigma Aldrich). 24 h after infection the cells were scraped into 500 µL of ice cold Buffer A (20 mM Tris, pH 7.5, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2) supplemented with 0.5% Triton X-100. Tris, pH 7.5, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2) supplemented with 0.5% Triton X-100. The cells were incubated on ice for 10 min and after that the nuclei were pelleted at 800 g for 10 min. The cells were incubated on ice for 10 min and after that the nuclei were pelleted at 800 g for 10 min. The cytoplasmic extract (C) was collected and centrifuged at 16,100 g for 15 min. To prepare the The cytoplasmic extract (C) was collected and centrifuged at 16,100 g for 15 min. To prepare the nuclear extract (N), the nuclear pellet was washed once with Buffer A + 0.5% Triton X-100 and twice nuclear extract (N), the nuclear pellet was washed once with Buffer A + 0.5% Triton X-100 and twice with Buffer A. The nuclei were suspended in 70 µl of Buffer B (20 mM¨Tris, pH 8.0, 500 mM¨NaCl, with Buffer A. The nuclei were suspended in 70 µl of Buffer B (20 mM¨Tris, pH 8.0, 500 mM¨NaCl, 2 mM¨EDTA, pH 8.0, 0.1% Igepal) and sonicated for 3 s. The nuclear proteins were collected after 2 mM¨EDTA, pH 8.0, 0.1% Igepal) and sonicated for 3 s. The nuclear proteins were collected after centrifugation at 16,100 g for 15 min. centrifugation at 16,100 g for 15 min.

2.7. Immunofluorescence Staining and Confocal Imaging 2.7. Immunofluorescence Staining and Confocal Imaging For immunofluorescence microscopy, A549 cells were grown on coverslips and infected at an MOI For immunofluorescence microscopy, A549 cells were grown on coverslips and infected at an MOI of 0.5 in the presence of TPCK-treated trypsin (5 µg/mL). At 20 h after infection, the cells were fixed of 0.5 in the presence of TPCK-treated trypsin (5 µg/mL). At 20 h after infection, the cells were fixed with ice cold methanol for 10 min at ´20 ˝C, permeabilized with 0.1% Triton X-100, and incubated with ice cold methanol for 10 min at ´20 ˝C, permeabilized with 0.1% Triton X-100, and incubated with guinea-pig anti-NS1 antibody, followed by AlexaFluor 488 goat anti-guinea pig IgG. CrkL was with guinea-pig anti-NS1 antibody, followed by AlexaFluor 488 goat anti-guinea pig IgG. CrkL was stained with mouse anti-CrkL antibody, followed by AlexaFluor 546 goat anti-mouse IgG. The cells stained with mouse anti-CrkL antibody, followed by AlexaFluor 546 goat anti-mouse IgG. The cells were then examined with Leica TCS SP8 confocal microscope. Channels were scanned sequentially. were then examined with Leica TCS SP8 confocal microscope. Channels were scanned sequentially. The mean intensities of the CrkL fluorescence signal in the nuclei were analyzed by using the open The mean intensities of the CrkL fluorescence signal in the nuclei were analyzed by using the open source software, FiJi distribution of ImageJ (Version 1.50b, NIH) [38]. source software, FiJi distribution of ImageJ (Version 1.50b, NIH) [38].

3. Results 3. Results

3.1. SH3 Binding-Competent NS1 Proteins Translocate Crk Proteins into the Nucleus 3.1. SH3 Binding-Competent NS1 Proteins Translocate Crk Proteins into the Nucleus To study the Crk/NS1 interaction in an infectious setting, we generated a set of recombinant To study the Crk/NS1 interaction in an infectious setting, we generated a set of recombinant viruses using a typical human IAV A/WSN/1933/H1N1 (A/WSN) as a background strain. viruses using a typical human IAV A/WSN/1933/H1N1 (A/WSN) as a background strain. These recombinant viruses are isogenic with wild-type A/WSN virus, except for the segment These recombinant viruses are isogenic with wild-type A/WSN virus, except for the segment 8 (NS segment), which encodes either the wild-type or a mutated NS1 from an avian IAV 8 (NS segment), which encodes either the wild-type or a mutated NS1 from an avian IAV A/Mallard/Netherlands/12/2000/H7N3 (A/Mallard) or a mutant construct of NS1 of A/WSN. To A/Mallard/Netherlands/12/2000/H7N3 (A/Mallard) or a mutant construct of NS1 of A/WSN. To generate an SH3 binding-incompetent mutant of the A/Mallard NS1, a K217E mutation was introduced generate an SH3 binding-incompetent mutant of the A/Mallard NS1, a K217E mutation was introduced into its NS1 sequence. Conversely, to engineer the naturally SH3 binding-incompetent A/WSN NS1 to into its NS1 sequence. Conversely, to engineer the naturally SH3 binding-incompetent A/WSN NS1 to become SH3 binding-competent, a T215P mutation was introduced into its NS1 sequence. Although become SH3 binding-competent, a T215P mutation was introduced into its NS1 sequence. Although not directly relevant for this study, it should be noted that the T215P mutation could also alter the not directly relevant for this study, it should be noted that the T215P mutation could also alter the phosphorylation pattern of NS1 as T215 has been reported as a functional phosphorylation site [39,40]. phosphorylation pattern of NS1 as T215 has been reported as a functional phosphorylation site [39,40]. The mutations made in the NS1 sequence do not affect the NS2/NEP open reading frame (ORF). The mutations made in the NS1 sequence do not affect the NS2/NEP open reading frame (ORF). The sequences of the relevant SH3-binding regions in the NS1 proteins of these viruses are shown in The sequences of the relevant SH3-binding regions in the NS1 proteins of these viruses are shown in Figure1A. Figure1A. To establish that the engineered mutations had the expected effects on the capacity of the To establish that the engineered mutations had the expected effects on the capacity of the corresponding A/Mallard and A/WSN NS1 proteins to interact with Crk proteins in IAV infected corresponding A/Mallard and A/WSN NS1 proteins to interact with Crk proteins in IAV infected cells, we immunoprecipitated endogenous CrkL (Figure1B,C) from mock infected or recombinant cells, we immunoprecipitated endogenous CrkL (Figure1B,C) from mock infected or recombinant virus-infected A549 cells and examined NS1 co-precipitation by Western blotting. As seen in Figure1B, virus-infected A549 cells and examined NS1 co-precipitation by Western blotting. As seen in Figure1B, while wild-type A/Mallard NS1 readily co-precipitated with CrkL, the NS1 mutant (K217E) did not while wild-type A/Mallard NS1 readily co-precipitated with CrkL, the NS1 mutant (K217E) did not associate with CrkL at detectable levels. Conversely, no association of wild-type A/WSN NS1 with associate with CrkL at detectable levels. Conversely, no association of wild-type A/WSN NS1 with CrkL could be detected, whereas efficient co-precipitation of the mutant NS1-T215P protein with a CrkL could be detected, whereas efficient co-precipitation of the mutant NS1-T215P protein with a restored Crk SH3-binding motif was observed (Figure1C). restored Crk SH3-binding motif was observed (Figure1C). Viruses 2016, 8, 101 5 of 15 Viruses 2016, 8, 101 5 of 15

Figure 1. A functional SH3 binding motif in the non-structural protein-1 (NS1) is required for interaction Figure 1. A functional SH3 binding motif in the non-structural protein-1 (NS1) is required for interaction with Crk-like adapter protein (CrkL) in influenza A virus (IAV)-infected cells. (A) The consensus with Crk-like adapter protein (CrkL) in influenza A virus (IAV)-infected cells. (A) The consensus sequence of class II SH3 binding motif, and its presence (+) or absence (´) in the C-terminal region sequence of class II SH3 binding motif, and its presence (+) or absence (´) in the C-terminal region (residues 212–217 shown) of NS1 proteins of the recombinant IAV strains used in this study. In SH3 (residues 212–217 shown) of NS1 proteins of the recombinant IAV strains used in this study. In SH3 binding consensus x indicates any residue, F a hydrophopic residue, and + a positively charged amino binding consensus x indicates any residue, F a hydrophopic residue, and + a positively charged amino acid, which for Crk-family SH3 domains is preferable a lysine residue; ( B,C) Co-immunoprecipation acid, which for Crk-family SH3 domains is preferable a lysine residue; ( B,C) Co-immunoprecipation of NS1 proteins with CrkL from lysates of A549 cells infected with recombinant A/WSN-based IAV of NS1 proteins with CrkL from lysates of A549 cells infected with recombinant A/WSN-based IAV strains expressing wild-type or mutant NS1 proteins derived from A/Mallard ( B) or A/WSN (C) for strains expressing wild-type or mutant NS1 proteins derived from A/Mallard ( B) or A/WSN (C) for 24 h at a multiplicity of infections (MOI) 2. Note that these NS1 proteins naturally differ in their 24 h at a multiplicity of infections (MOI) 2. Note that these NS1 proteins naturally differ in their SH3 binding capacity, and the mutations introduced in them thus have opposite effects. NS1 and SH3 binding capacity, and the mutations introduced in them thus have opposite effects. NS1 and nucleoprotein (NP) blots from whole cell extracts (WCE) before anti-CrkL immunoprecipitation are nucleoprotein (NP) blots from whole cell extracts (WCE) before anti-CrkL immunoprecipitation are shown to control equal infection of the cells by the different viruses. shown to control equal infection of the cells by the different viruses.

Next, we analyzed the localization of NS1 and Crk proteins in the infected cells by Next, we analyzed the localization of NS1 and Crk proteins in the infected cells by immunofluorescence staining and confocal imaging. A549 cells were infected with an MOI of immunofluorescence staining and confocal imaging. A549 cells were infected with an MOI of 0.5 with A/WSN-NS1Mallard(wt) or A/WSN-NS1Mallard(K217E) recombinant viruses and the cells were 0.5 with A/WSN-NS1Mallard(wt) or A/WSN-NS1Mallard(K217E) recombinant viruses and the cells were fixed 20 h later. CrkL was localized mainly in the cytoplasm in mock-infected cells, and only faint fixed 20 h later. CrkL was localized mainly in the cytoplasm in mock-infected cells, and only faint staining was observed in the nucleus (Figure2A, top row). In the infected cells, both the WT and staining was observed in the nucleus (Figure2A, top row). In the infected cells, both the WT and the K217E-mutant NS1 proteins were predominantly localized in the nucleus (Figure2A, in green). the K217E-mutant NS1 proteins were predominantly localized in the nucleus (Figure2A, in green). Strikingly, in cells infected with A/WSN-NS1Mallard(wt) CrkL was found to mainly co-localize with NS1 Strikingly, in cells infected with A/WSN-NS1Mallard(wt) CrkL was found to mainly co-localize with NS1 in the nucleus (Figure2A, middle row), whereas in cells infected with A/WSN-NS1 Mallard(K217E) the in the nucleus (Figure2A, middle row), whereas in cells infected with A/WSN-NS1 Mallard(K217E) the distribution of CrkL was indistinguishable from its predominantly cytoplasmic localization pattern in distribution of CrkL was indistinguishable from its predominantly cytoplasmic localization pattern in mock-infected cells (Figure2A, bottom row). Very similar differential distribution was also observed mock-infected cells (Figure2A, bottom row). Very similar differential distribution was also observed for Crk when examined by immunostaining with an antibody that detects both CrkI and CrkII (data for Crk when examined by immunostaining with an antibody that detects both CrkI and CrkII (data not shown). To more formally establish this effect, the mean intensity of CrkL-fluorescence signal not shown). To more formally establish this effect, the mean intensity of CrkL-fluorescence signal in NS1-positive nuclei was quantified from 50 individual cells infected with A/WSN-NS1Mallard(wt), in NS1-positive nuclei was quantified from 50 individual cells infected with A/WSN-NS1Mallard(wt), Mallard(K217E) 1 Mallard(K217E) 1 A/WSN-NS1 , or mock-infected cells (Figure2A, right panel). When the mean intensities A/WSN-NS1 , or mock-infected cells (Figure2A, right panel). When the mean intensities Viruses 2016, 8, x 6 of 14 Viruses 2016, 8, x 6 of 14 mean intensities of CrkL immunostaining in these nuclei were normalized to the value of the mean intensities of CrkL immunostaining in these nuclei were normalized to the value of the mock-infected cells, a robust and highly significant nuclear translocation of CrkL by SH3 mock-infected cells, a robust and highly significant nuclear translocation of CrkL by SH3 binding-competent but not by SH3 binding-incompetent NS1 could be demonstrated. binding-competent but not by SH3 binding-incompetent NS1 could be demonstrated. Viruses 2016, 8, 101 6 of 15 Viruses 2016, 8, 101 6 of 15 When the kinetics of nuclear translocation of the Crk proteins was examined in more detail, we When the kinetics of nuclear translocation of the Crk proteins was examined in more detail, we could observe first signs of nuclear accumulation of the Crk and CrkL at 6 h post-infection (p.i.) could observe first signs of nuclear accumulation of the Crk and CrkL at 6 h post-infection (p.i.) ofcoinciding CrkL immunostaining with the nuclei in becoming these nuclei clearly were positive normalized for NS1 to staining the value (Figure of the 2B). mock-infected At 8 h p.i. nuclear cells, a ofcoinciding CrkL immunostaining with the nuclei in becoming these nuclei clearly were positive normalized for NS1 to staining the value (Figure of the 2B). mock-infected At 8 h p.i. nuclear cells, a robustaccumulation and highly of Crk significant proteins nuclear was already translocation prominent, of CrkL and by at SH3 12 h binding-competent p.i. Crk/CrL localization but not seemed by SH3 robustaccumulation and highly of Crk significant proteins nuclear was already translocation prominent, of CrkL and by at SH3 12 h binding-competent p.i. Crk/CrL localization but not seemed by SH3 binding-incompetentalready complete showing NS1 could a patter ben demonstrated. that looked identical to the 20 h p.i. time point shown in Figure binding-incompetentalready complete showing NS1 could a patter ben demonstrated. that looked identical to the 20 h p.i. time point shown in Figure 2A. 2A.

FigureFigure 2. 2. InfectionInfection of of cells cells with with IAV IAV expressing SH3 binding-competent binding-competent NS1 causes causes nuclear nuclear FigureFigure 2. 2. InfectionInfection of of cells cells with with IAV IAV expressing SH3 binding-competent binding-competent NS1 causes causes nuclear nuclear relocalizationrelocalization of of CrkL. CrkL. (A (A) )Immunofluorescence Immunofluorescence staining staining of ofNS1 NS1 and and CrkL CrkL in A549 in A549 cells cellsthat w thatere relocalizationrelocalization of of CrkL. CrkL. (A (A) )Immunofluorescence Immunofluorescence staining staining of ofNS1 NS1 and and CrkL CrkL in A549 in A549 cells cellsthat w thatere weremock- mock-infectedinfected (upper (upper panel) panel) or or infected infected with with A/WSN-NS1 A/WSN-NS1Mallard(wt)Mallard(wt) (middle(middle panel) panel) or or weremock- mock-infectedinfected (upper (upper panel) panel) or or infected infected with with A/WSN-NS1 A/WSN-NS1Mallard(wt)Mallard(wt) (middle(middle panel) panel) or or A/WSN-NS1A/WSN-NS1Mallard(K217E)Mallard(K217E) (bottom(bottom panel) panel) for 20 for h 20at a h MOI at a 0.5. MOI The 0.5. nuclei The were nuclei visualized were visualized by staining by A/WSN-NS1A/WSN-NS1Mallard(K217E)Mallard(K217E) (bottom(bottom panel) panel) for 20 for h 20at a h MOI at a 0.5. MOI The 0.5. nuclei The were nuclei visualized were visualized by staining by stainingwith Hoechst. with Hoechst.The mean The intensity mean intensityof CrkL offluorescence CrkL fluorescence in the nucle in thei was nuclei quantified was quantified from 50 fromcells stainingwith Hoechst. with Hoechst.The mean The intensity mean intensityof CrkL offluorescence CrkL fluorescence in the nucle in thei was nuclei quantified was quantified from 50 fromcells 50infected cells infected with A/WSN with- A/WSN-NS1NS1Mallard(wt) orMallard(wt) A/WSN-orNS1 A/WSN-NS1Mallard(K217E) thatMallard(K217E) also stainedthat positive also stained for positive positive for 50infected cells infected with A/WSN with- A/WSN-NS1NS1Mallard(wt) orMallard(wt) A/WSN-orNS1 A/WSN-NS1Mallard(K217E) thatMallard(K217E) also stainedthat positive also stained for positive positive for forNS1, positive and was for NS1, normalized and was tonormalized the mean to fluorescencethe mean fluorescence intensity intensity of CrkL of immunostaining CrkL immunostaining of 50 forNS1, positive and was for NS1, normalized and was tonormalized the mean to fluorescencethe mean fluorescence intensity intensity of CrkL of immunostaining CrkL immunostaining of 50 ofmock 50 mock-infected-infected cells. cells.The stand Theard standard error is error presented is presented in the infigure. the figure. The statistical The statistical significance significance of the ofmock 50 mock-infected-infected cells. cells.The stand Theard standard error is error presented is presented in the infigure. the figure. The statistical The statistical significance significance of the ofdifferences the differences was determined was determined by Student by Student′s t-test1 (*s t-testp < 0.001) (* p ;< (B 0.001);) A549 ( Bcells) A549 were cells infected were infectedwith A/WSN with- ofdifferences the differences was determined was determined by Student by Student′s t-test1 (*s t-testp < 0.001) (* p ;< (B 0.001);) A549 ( Bcells) A549 were cells infected were infectedwith A/WSN with- A/WSN-NS1NS1Mallard(wt) forMallard(wt) differentfor time different points time at a points MOI 0.5. at a The MOI localization 0.5. The localization of CrkL was of CrkL scored was from scored 100 fromcells A/WSN-NS1NS1Mallard(wt) forMallard(wt) differentfor time different points time at a points MOI 0.5. at a The MOI localization 0.5. The localization of CrkL was of CrkL scored was from scored 100 fromcells 100as a cellscytoplasmic, as a cytoplasmic, an intermediate an intermediate,, or a nuclear or a pattern. nuclear pattern. 100as a cellscytoplasmic, as a cytoplasmic, an intermediate an intermediate,, or a nuclear or a pattern. nuclear pattern.

Viruses 2016, 8, x 7 of 14 Viruses 2016, 8, x 7 of 14

To extend and support these imaging studies by using a biochemical approach, we prepared To extend and support these imaging studies by using a biochemical approach, we prepared cytoplasmic and nuclear fractions of cells infected with recombinant viruses and compared the cytoplasmic and nuclear fractions of cells infected with recombinant viruses and compared the presence of Crk proteins and wild-type or mutant NS1 proteins in these fractions (Figure 3A,B). The presence of Crk proteins and wild-type or mutant NS1 proteins in these fractions (Figure 3A,B). The quality and purity of the nuclear and cytoplasmic protein fractions obtained from these cells were quality and purity of the nuclear and cytoplasmic protein fractions obtained from these cells were establishedViruses 2016 by, 8, 101 Western blotting of these preparations using antibodies against anti-α7- oftubulin 15 establishedViruses 2016 by, 8, 101 Western blotting of these preparations using antibodies against anti-α7- oftubulin 15 (a cytoplasmic marker) and anti-histone H3 (a nuclear marker). (a cytoplasmic marker) and anti-histone H3 (a nuclear marker). As expected,When the NS1 kinetics protein of nuclear was seen translocation mainly in ofthe the nuclear Crk proteins fractions was regardless examined of in the more recombinant detail, As expected,When the NS1 kinetics protein of nuclear was seen translocation mainly in ofthe the nuclear Crk proteins fractions was regardless examined of in the more recombinant detail, viruswe that could was observe used to first infect signs these of nuclear cells. accumulationThe nuclear fractions of the Crk of and mock CrkL-infected at 6 h post-infection cells did not (p.i.) contain viruswe that could was observe used to first infect signs these of nuclear cells. accumulationThe nuclear fractions of the Crk of and mock CrkL-infected at 6 h post-infection cells did not (p.i.) contain detectablecoinciding amounts with theof CrkI, nuclei CrkII becoming, or CrkL clearly proteins positive (Figure for NS1 3A staining,B), whereas (Figure strong2B). At signals 8 h p.i. nuclearof expected detectablecoinciding amounts with theof CrkI, nuclei CrkII becoming, or CrkL clearly proteins positive (Figure for NS1 3A staining,B), whereas (Figure strong2B). At signals 8 h p.i. nuclearof expected size accumulation for these proteins of Crk proteins were wasobserved already in prominent, the cytoplasmic and at 12 h fractions. p.i. Crk/CrL Nuclear localization vs. cytoplasmic seemed size accumulation for these proteins of Crk proteins were wasobserved already in prominent, the cytoplasmic and at 12 h fractions. p.i. Crk/CrL Nuclear localization vs. cytoplasmic seemed fractioalreadynation complete of Crk showingproteins a patternderived that from looked cells identical infected to thewith 20 hviruses p.i. time expressing point shown an in SH3 Figure binding2A. - fractioalreadynation complete of Crk showingproteins a patternderived that from looked cells identical infected to thewith 20 hviruses p.i. time expressing point shown an in SH3 Figure binding2A. - To extend and support these imaging studies by using a biochemical approach, we prepared To extend and support these imaging studies by using a biochemical approach, we prepared incompetent version of NS1 (A/WSN-NS1WSN(wt) and A/WSN-NS1Mallard(K217E)) was identical with that incompetent version of NS1 (A/WSN-NS1WSN(wt) and A/WSN-NS1Mallard(K217E)) was identical with that cytoplasmic and nuclear fractions of cells infected with recombinant viruses and compared the presence cytoplasmic and nuclear fractions of cells infected with recombinant viruses and compared the presence observed with mock-infected cells (Figure 3A,B). In sharp contrast, in cells infected with viruses observed with mock-infected cells (Figure 3A,B). In sharp contrast, in cells infected with viruses of Crk proteins and wild-type or mutant NS1 proteins in these fractions (Figure3A,B). The quality and of Crk proteins and wild-type or mutant NS1 proteins in these fractions (Figure3A,B). The quality and WSN(T215P) Mallard(wt) WSN(T215P) Mallard(wt) havingpurity an of SH3 the nuclear binding and-competent cytoplasmic NS1 protein (A/WSN fractions-NS1 obtained from and these A/WSN cells were-NS1 established) all by Crk havingpurity an of SH3 the nuclear binding and-competent cytoplasmic NS1 protein (A/WSN fractions-NS1 obtained from and these A/WSN cells were-NS1 established) all by Crk proteinsWestern could blotting be of abundantly these preparations detected using in antibodies the nuclear against fractions, anti-α-tubulin especially (a cytoplasmic CrkL marker) becoming proteinsWestern could blotting be of abundantly these preparations detected using in antibodies the nuclear against fractions, anti-α-tubulin especially (a cytoplasmic CrkL marker) becoming predominantlyand anti-histone nuclear H3 (a(Figure nuclear 3A marker).,B). predominantlyand anti-histone nuclear H3 (a(Figure nuclear 3A marker).,B).

FigureFigure 3. Nuclear 3. Nuclear translocation translocation of ofCrkI, CrkI, CrkII, CrkII, and CrkL byby SH3 SH3 binding-competent binding-competent NS1 NS1 proteins proteins FigureFigure 3. Nuclear 3. Nuclear translocation translocation of ofCrkI, CrkI, CrkII, CrkII, and CrkL byby SH3 SH3 binding-competent binding-competent NS1 NS1 proteins proteins demonstrateddemonstrated by subcellular by subcellular fractionation. fractionation. (A (A)) Western Western blotblot analysis analysis of of cytoplasmic cytoplasmic (C) ( andC) and nuclear nuclear demonstrateddemonstrated by subcellular by subcellular fractionation. fractionation. (A (A)) Western Western blotblot analysis analysis of of cytoplasmic cytoplasmic (C) ( andC) and nuclear nuclear extractsextracts (N) (preparedN) prepared from from A549 A549 cells cells that that were were mock-infectedmock-infected ( MOCK(MOCK) or) or infected infected for 24for h24 with h with extractsextracts (N) (preparedN) prepared from from A549 A549 cells cells that that were were mock-infectedmock-infected ( MOCK(MOCK) or) or infected infected for 24for h24 with h with recombinantrecombinant A/WSN A/WSN containing containing either either the the wild wild-type-type ((WTWT)) or or the the K217E K217E mutant mutant NS1 NS1 from from A/Mallard A/Mallard recombinantrecombinant A/WSN A/WSN containing containing either either the the wild wild-type-type ((WTWT)) or or the the K217E K217E mutant mutant NS1 NS1 from from A/Mallard A/Mallard virusvirus at an at MOI an MOI 2. In 2. addition In addition to toantibodies antibodies against against the Crk-familyCrk-family proteins proteins and and NS1, NS1 the, the blotted blotted A549 A549 virusvirus at an at MOI an MOI 2. In 2. addition In addition to toantibodies antibodies against against the Crk-familyCrk-family proteins proteins and and NS1, NS1 the, the blotted blotted A549 A549 fractions were also probed with antibodies against Histone H3 and α-tubulin to confirm successful fractions were also probed with antibodies against Histone H3 and α-tubulin to confirm successful fractions were also probed with antibodies against Histone H3 and α-tubulin to confirm successful fractions were also probed with antibodies against Histone H3 and α-tubulin to confirm successful separation of nuclear and cytoplasmic fractions. In addition, unfractionated whole cell extracts (WCE) separation of nuclear and cytoplasmic fractions. In addition, unfractionated whole cell extracts (WCE) separation of nuclear and cytoplasmic fractions. In addition, unfractionated whole cell extracts (WCE) separation of nuclear and cytoplasmic fractions. In addition, unfractionated whole cell extracts (WCE) of the infected cells were Western blotted with anti-NS1 and anti-NP antibodies to confirm uniform of the infected cells were Western blotted with anti-NS1 and anti-NP antibodies to confirm uniform of theinfection infected of cells cells were by the Western different blotted viruses; with (B) Sameanti-NS1 as (A and) except anti- thatNP theantibodies cells were to infectedconfirm withuniform of theinfection infected of cells cells were by the Western different blotted viruses; with (B) Sameanti-NS1 as (A and) except anti- thatNP theantibodies cells were to infectedconfirm withuniform infectionrecombinant of cells A/WSN by the virusdifferent carrying viruses wild-type; (B) Same (WT) oras the(A T215P) except mutant that NS1the fromcells A/WSN.were infected with infectionrecombinant of cells A/WSN by the virusdifferent carrying viruses wild-type; (B) Same (WT) oras the(A T215P) except mutant that NS1the fromcells A/WSN.were infected with recombinant A/WSN virus carrying wild-type (WT) or the T215P mutant NS1 from A/WSN. recombinant A/WSN virus carrying wild-type (WT) or the T215P mutant NS1 from A/WSN.

3.2 NS1-Induced PI3K-Activation does not Depend on Crk Relocalization into the Nucleus 3.2 NS1-Induced PI3K-Activation does not Depend on Crk Relocalization into the Nucleus Our previous studies have shown that simultaneous recruitment of Crk proteins by NS1 Our previous studies have shown that simultaneous recruitment of Crk proteins by NS1 substantially potentiates NS1-induced activation of PI3-kinase pathway [21, 22]. While these substantially potentiates NS1-induced activation of PI3-kinase pathway [21, 22]. While these signaling interactions would be expected to take place in the cytoplasm, it is nevertheless possible signaling interactions would be expected to take place in the cytoplasm, it is nevertheless possible

Viruses 2016, 8, 101 8 of 15 Viruses 2016, 8, 101 8 of 15

As expected, NS1 protein was seen mainly in the nuclear fractions regardless of the recombinant As expected, NS1 protein was seen mainly in the nuclear fractions regardless of the recombinant virus that was used to infect these cells. The nuclear fractions of mock-infected cells did not contain virus that was used to infect these cells. The nuclear fractions of mock-infected cells did not contain detectable amounts of CrkI, CrkII, or CrkL proteins (Figure3A,B), whereas strong signals of expected detectable amounts of CrkI, CrkII, or CrkL proteins (Figure3A,B), whereas strong signals of expected size for these proteins were observed in the cytoplasmic fractions. Nuclear vs. cytoplasmic fractionation size for these proteins were observed in the cytoplasmic fractions. Nuclear vs. cytoplasmic fractionation of Crk proteins derived from cells infected with viruses expressing an SH3 binding-incompetent version of Crk proteins derived from cells infected with viruses expressing an SH3 binding-incompetent version of NS1 (A/WSN-NS1WSN(wt) and A/WSN-NS1Mallard(K217E)) was identical with that observed with of NS1 (A/WSN-NS1WSN(wt) and A/WSN-NS1Mallard(K217E)) was identical with that observed with mock-infected cells (Figure3A,B). In sharp contrast, in cells infected with viruses having an SH3 mock-infected cells (Figure3A,B). In sharp contrast, in cells infected with viruses having an SH3 binding-competent NS1 (A/WSN-NS1WSN(T215P) and A/WSN-NS1Mallard(wt)) all Crk proteins could binding-competent NS1 (A/WSN-NS1WSN(T215P) and A/WSN-NS1Mallard(wt)) all Crk proteins could be abundantly detected in the nuclear fractions, especially CrkL becoming predominantly nuclear be abundantly detected in the nuclear fractions, especially CrkL becoming predominantly nuclear (Figure3A,B). (Figure3A,B).

3.2. NS1-Induced PI3K-Activation does not Depend on Crk Relocalization into the Nucleus 3.2. NS1-Induced PI3K-Activation does not Depend on Crk Relocalization into the Nucleus Our previous studies have shown that simultaneous recruitment of Crk proteins by NS1 Our previous studies have shown that simultaneous recruitment of Crk proteins by NS1 substantially potentiates NS1-induced activation of PI3-kinase pathway [21,22]. While these signaling substantially potentiates NS1-induced activation of PI3-kinase pathway [21,22]. While these signaling interactions would be expected to take place in the cytoplasm, it is nevertheless possible that interactions would be expected to take place in the cytoplasm, it is nevertheless possible that subsequent nuclear transit of the bulk of cellular Crk proteins by NS1 could somehow contribute to subsequent nuclear transit of the bulk of cellular Crk proteins by NS1 could somehow contribute to the observed PI3K superactivation. the observed PI3K superactivation. To address this possibility we generated a mutant NS1 protein that remains predominantly in the To address this possibility we generated a mutant NS1 protein that remains predominantly in the cytoplasm (NS1-Cyto). This was achieved by mutating the N-terminal NLS1 of A/Mallard NS1 (this cytoplasm (NS1-Cyto). This was achieved by mutating the N-terminal NLS1 of A/Mallard NS1 (this strain does not contain NLS2) at the critical basic residues (R38A,R41A) combined with the addition strain does not contain NLS2) at the critical basic residues (R38A,R41A) combined with the addition of a strong heterologous nuclear export signal (NES) from mitogen-activated protein kinase kinase-1 of a strong heterologous nuclear export signal (NES) from mitogen-activated protein kinase kinase-1 (MAPKK1) [41]. (MAPKK1) [41]. The localization of NS1-Cyto was compared with wild-type A/Mallard NS1 by transient The localization of NS1-Cyto was compared with wild-type A/Mallard NS1 by transient transfection of red fluorescent fusion protein (mCherry) derivatives of these NS1 proteins. Similar to transfection of red fluorescent fusion protein (mCherry) derivatives of these NS1 proteins. Similar to the NS1 immunostaining in IAV-infected cells, in virtually all productively NS1-transfected cells (96%; the NS1 immunostaining in IAV-infected cells, in virtually all productively NS1-transfected cells (96%; of 100 cells counted) the red fluorescence of wild-type NS1 showed a distinctly nuclear localization of 100 cells counted) the red fluorescence of wild-type NS1 showed a distinctly nuclear localization pattern as illustrated in Figure4A (upper panel). By contrast, only 1% of cells transfected with pattern as illustrated in Figure4A (upper panel). By contrast, only 1% of cells transfected with NS1-Cyto showed such a nuclear fluorescence, and in almost all (92%) of these cells the nuclei were NS1-Cyto showed such a nuclear fluorescence, and in almost all (92%) of these cells the nuclei were devoid of NS1 signal and appeared as dark areas inside cytoplasmic red fluorescence (Figure4A, devoid of NS1 signal and appeared as dark areas inside cytoplasmic red fluorescence (Figure4A, lower panel), thus establishing the success of our double mutation approach to generate an NS1 lower panel), thus establishing the success of our double mutation approach to generate an NS1 mutant restricted to a cytoplasmic localization. Co-transfection of a vector expressing CrkL tagged mutant restricted to a cytoplasmic localization. Co-transfection of a vector expressing CrkL tagged with eGFP recapitulated our results obtained by infection with recombinant IAV variants, showing a with eGFP recapitulated our results obtained by infection with recombinant IAV variants, showing a prominently nuclear green fluorescence that faithfully co-localized with wild-type NS1. Conversely, in prominently nuclear green fluorescence that faithfully co-localized with wild-type NS1. Conversely, in NS1-Cyto-transfected cells also CrkL fluorescence was found predominantly in the cytoplasm (see NS1-Cyto-transfected cells also CrkL fluorescence was found predominantly in the cytoplasm (see Figure4B for statistics of the observed NS1 and CrkL localization patterns). When eGFP-CrkL was Figure4B for statistics of the observed NS1 and CrkL localization patterns). When eGFP-CrkL was transfected alone (data not shown), a localization pattern similar to that observed for endogenous transfected alone (data not shown), a localization pattern similar to that observed for endogenous CrkL by immunostaining (Figure2A). CrkL by immunostaining (Figure2A). When the capacity of NS1-Cyto to trigger PI3K-activation in transfected cells was compared with When the capacity of NS1-Cyto to trigger PI3K-activation in transfected cells was compared with that of wild-type A/Mallard NS1, a similar increase in the phosphorylation of Akt, a downstream that of wild-type A/Mallard NS1, a similar increase in the phosphorylation of Akt, a downstream effector of the PI3K cascade was observed (Figure4C). Thus, while recruitment of Crk proteins by NS1 effector of the PI3K cascade was observed (Figure4C). Thus, while recruitment of Crk proteins by NS1 is required for the enhancement PI3K-activation [21,22], NS1-mediated nuclear translocation of Crk is required for the enhancement PI3K-activation [21,22], NS1-mediated nuclear translocation of Crk is not. Likewise, it can be concluded that while the cytoplasmic interaction between NS1 and Crk is is not. Likewise, it can be concluded that while the cytoplasmic interaction between NS1 and Crk is sufficient to potentiate PI3K activation, nuclear targeting of Crk cannot be triggered by a contact with sufficient to potentiate PI3K activation, nuclear targeting of Crk cannot be triggered by a contact with NS1 in the cytoplasm, but indeed physically depends on the nuclear entry of Crk-NS1-complex driven NS1 in the cytoplasm, but indeed physically depends on the nuclear entry of Crk-NS1-complex driven by the NLS of NS1. by the NLS of NS1. Viruses 2016, 8, x 8 of 14 Viruses 2016, 8, x 8 of 14 that subsequent nuclear transit of the bulk of cellular Crk proteins by NS1 could somehow contribute that subsequent nuclear transit of the bulk of cellular Crk proteins by NS1 could somehow contribute to the observed PI3K superactivation. to the observed PI3K superactivation. To address this possibility we generated a mutant NS1 protein that remains predominantly in To address this possibility we generated a mutant NS1 protein that remains predominantly in the cytoplasm (NS1-Cyto). This was achieved by mutating the N-terminal NLS1 of A/Mallard NS1 the cytoplasm (NS1-Cyto). This was achieved by mutating the N-terminal NLS1 of A/Mallard NS1 (this strain does not contain NLS2) at the critical basic residues (R38A,R41A) combined with the (this strain does not contain NLS2) at the critical basic residues (R38A,R41A) combined with the addition of a strong heterologous nuclear export signal (NES) from mitogen-activated protein kinase addition of a strong heterologous nuclear export signal (NES) from mitogen-activated protein kinase kinase-1 (MAPKK1) [41]. kinase-1 (MAPKK1) [41]. The localization of NS1-Cyto was compared with wild-type A/Mallard NS1 by transient The localization of NS1-Cyto was compared with wild-type A/Mallard NS1 by transient transfection of red fluorescent fusion protein (mCherry) derivatives of these NS1 proteins. Similar to transfection of red fluorescent fusion protein (mCherry) derivatives of these NS1 proteins. Similar to the NS1 immunostaining in IAV-infected cells, in virtually all productively NS1-transfected cells the NS1 immunostaining in IAV-infected cells, in virtually all productively NS1-transfected cells (96%; of 100 cells counted) the red fluorescence of wild-type NS1 showed a distinctly nuclear (96%; of 100 cells counted) the red fluorescence of wild-type NS1 showed a distinctly nuclear localization pattern as illustrated in Figure 4A (upper panel). By contrast, only 1% of cells transfected localization pattern as illustrated in Figure 4A (upper panel). By contrast, only 1% of cells transfected with NS1-Cyto showed such a nuclear fluorescence, and in almost all (92%) of these cells the nuclei with NS1-Cyto showed such a nuclear fluorescence, and in almost all (92%) of these cells the nuclei were devoid of NS1 signal and appeared as dark areas inside cytoplasmic red fluorescence (Figure were devoid of NS1 signal and appeared as dark areas inside cytoplasmic red fluorescence (Figure 4A, lower panel), thus establishing the success of our double mutation approach to generate an NS1 4A, lower panel), thus establishing the success of our double mutation approach to generate an NS1 mutant restricted to a cytoplasmic localization. Co-transfection of a vector expressing CrkL tagged mutant restricted to a cytoplasmic localization. Co-transfection of a vector expressing CrkL tagged with eGFP recapitulated our results obtained by infection with recombinant IAV variants, showing with eGFP recapitulated our results obtained by infection with recombinant IAV variants, showing a prominently nuclear green fluorescence that faithfully a prominently nuclear green fluorescence that faithfully co-localized with wild-type NS1. Conversely, in NS1-Cyto-transfected cells also CrkL fluorescence co-localized with wild-type NS1. Conversely, in NS1-Cyto-transfected cells also CrkL fluorescence was found predominantly in the cytoplasm (see Figure 4B for statistics of the observed NS1 and CrkL was found predominantly in the cytoplasm (see Figure 4B for statistics of the observed NS1 and CrkL

Viruseslocalization2016, 8, 101 patterns). When eGFP-CrkL was transfected alone (data not shown), a localization9 of 15 Viruseslocalization2016, 8, 101 patterns). When eGFP-CrkL was transfected alone (data not shown), a localization9 of 15 pattern similar to that observed for endogenous CrkL by immunostaining (Figure 2A). pattern similar to that observed for endogenous CrkL by immunostaining (Figure 2A).

Figure 4. NS1NS1-mediated-mediated Crk Crk relocalization relocalization is in isdependent independent of NS1 of NS1-activated-activated PI3K PI3K-signaling.-signaling. (A) Figure 4. NS1NS1-mediated-mediated Crk Crk relocalization relocalization is in isdependent independent of NS1 of NS1-activated-activated PI3K PI3K-signaling.-signaling. (A) (FluoresenceA) Fluoresence microscopy microscopy imaging imaging of of Huh7 Huh7 cells cells co co-transfected-transfected with with eGFP eGFP-fused-fused CrkL CrkL (green (green (FluoresenceA) Fluoresence microscopy microscopy imaging imaging of of Huh7 Huh7 cells cells co co-transfected-transfected with with eGFP eGFP-fused-fused CrkL CrkL (green (green fluorescense) together with mCherry-fusion protein (red fluorescence) of wild-type A/Mallard NS1 fluorescense) together with mCherry-fusion protein (red fluorescence) of wild-type A/Mallard NS1

(WT) or its dominantly cytoplasmic mutant NS1-Cyto; (B) Localization of NS1 and CrkL was examined (WT) or its dominantly cytoplasmic mutant NS1-Cyto; (B) Localization of NS1 and CrkL was examined in 100 cells from (A) and the observed fluorescence patterns were scored as nuclear, intermediate, in 100 cells from (A) and the observed fluorescence patterns were scored as nuclear, intermediate, or cytoplasmic; (C) PI3K-activation by wild-type and mutant version of NS1 revealed by Akt or cytoplasmic; (C) PI3K-activation by wild-type and mutant version of NS1 revealed by Akt phosphorylation. Huh7 cells were transiently transfected with a vector expressing the indicated phosphorylation. Huh7 cells were transiently transfected with a vector expressing the indicated NS1 variants, and 48 h later examined by Western blotting with antibodies against phospho-Akt (pAkt), NS1 variants, and 48 h later examined by Western blotting with antibodies against phospho-Akt (pAkt), NS1, and α-tubulin. NS1, and α-tubulin.

3.3. A Change in Nuclear Protein Tyrosine Phosphorylation after NS1-Mediated Nuclear Re-Localization of Crk 3.3. A Change in Nuclear Protein Tyrosine Phosphorylation after NS1-Mediated Nuclear Re-Localization of Crk Crk proteins interact with many tyrosine phosphorylated proteins as well as tyrosine kinases [25], Crk proteins interact with many tyrosine phosphorylated proteins as well as tyrosine kinases [25], and upon the original discovery of the viral Crk oncogene (v-Crk) an increase in cellular protein and upon the original discovery of the viral Crk oncogene (v-Crk) an increase in cellular protein tyrosine phosphorylation was described as a hallmark of Crk-mediated malignant transformation [42]. tyrosine phosphorylation was described as a hallmark of Crk-mediated malignant transformation [42]. To study whether NS1-mediated nuclear translocation of Crk proteins in IAV-infected cells would lead To study whether NS1-mediated nuclear translocation of Crk proteins in IAV-infected cells would lead to any functional consequences, we compared the patterns of protein tyrosine phosphorylation in to any functional consequences, we compared the patterns of protein tyrosine phosphorylation in nuclear extracts of A549 cells that were mock-infected or infected for 24 h with IAV expressing nuclear extracts of A549 cells that were mock-infected or infected for 24 h with IAV expressing NS1 proteins either capable (A/WSN-NS1Mallard(wt) and A/WSN-NS1WSN(T215P)) or not capable NS1 proteins either capable (A/WSN-NS1Mallard(wt) and A/WSN-NS1WSN(T215P)) or not capable (A/WSN-NS1WSN(wt) and A/WSN-NS1Mallard(K217E)) for binding and nuclear targeting of Crk. (A/WSN-NS1WSN(wt) and A/WSN-NS1Mallard(K217E)) for binding and nuclear targeting of Crk. To enhance the accumulation of phosphotyrosine-modified proteins, the cells were treated for 10 min To enhance the accumulation of phosphotyrosine-modified proteins, the cells were treated for 10 min with the phosphotyrosine phosphatase-inhibitor pervanadate before they were fractionated into with the phosphotyrosine phosphatase-inhibitor pervanadate before they were fractionated into nuclear and cytoplasmic extracts that were subjected to Western blotting with an anti-phosphotyrosine nuclear and cytoplasmic extracts that were subjected to Western blotting with an anti-phosphotyrosine (anti-pTyr) antibody (Figure5). Successful subcellular fractionation was confirmed by probing with (anti-pTyr) antibody (Figure5). Successful subcellular fractionation was confirmed by probing with Viruses 2016, 8, 101 10 of 15 Viruses 2016, 8, 101 10 of 15 antibodies against prototypic nuclear and cytoplasmic proteins, and uniform infection of the cells was antibodies against prototypic nuclear and cytoplasmic proteins, and uniform infection of the cells was demonstrated by probing unfractionated lysates of these cells with antibodies against IAV NS1 and NP. demonstrated by probing unfractionated lysates of these cells with antibodies against IAV NS1 and NP. While the nuclear extracts of cells infected with viruses expressing NS1 proteins lacking Crk binding While the nuclear extracts of cells infected with viruses expressing NS1 proteins lacking Crk binding activity did not differ from mock-infected cells in their patterns of tyrosine phosphorylated proteins, activity did not differ from mock-infected cells in their patterns of tyrosine phosphorylated proteins, a prominent new phosphotyrosine-containing protein with a MW of about 135 kDa appeared in the a prominent new phosphotyrosine-containing protein with a MW of about 135 kDa appeared in the nuclear extracts of cells infected with A/WSN-NS1Mallard(wt) or A/WSN-NS1WSN(T215P) (Figure5, nuclear extracts of cells infected with A/WSN-NS1Mallard(wt) or A/WSN-NS1WSN(T215P) (Figure5, pointed with arrows). Thus, we conclude that Crk proteins translocated into the nucleus upon pointed with arrows). Thus, we conclude that Crk proteins translocated into the nucleus upon IAV infection via their binding to NS1 can reprogram cellular signaling pathways in the nucleus as IAV infection via their binding to NS1 can reprogram cellular signaling pathways in the nucleus as Virusesevidenced 2016, 8 by, x altered nuclear protein tyrosine phosphorylation. 10 of 14 Virusesevidenced 2016, 8 by, x altered nuclear protein tyrosine phosphorylation. 10 of 14

Figure 5. NuclearNuclear targeting targeting of of Crk Crk by by NS1 NS1 causes causes a a change change in in the nuclear protein tyrosine Figure 5. NuclearNuclear targeting targeting of of Crk Crk by by NS1 NS1 causes causes a a change change in in the nuclear protein tyrosine phosphorylphosphorylationation pattern pattern of of IAV IAV-infected-infected cells. cells. A549 A549 cells cells were were infected infected with with recombinant recombinant viruses viruses as phosphorylphosphorylationation pattern pattern of of IAV IAV-infected-infected cells. cells. A549 A549 cells cells were were infected infected with with recombinant recombinant viruses viruses as indicatedas indicated for for24 h 24 at h a at MOI a MOI 2, and 2, andtreated treated with with pervanadate pervanadate for 10 for min 10 minbefore before cytoplasmic cytoplasmic (C) and (C) indicatedas indicated for for24 h 24 at h a at MOI a MOI 2, and 2, andtreated treated with with pervanadate pervanadate for 10 for min 10 minbefore before cytoplasmic cytoplasmic (C) and (C) nuclearand nuclear (N ()N ) extracts extracts were were prepared. prepared. The extracts The were extracts probed with were an anti-phosphotyrosine probed with an nuclearand nuclear (N ()N ) extracts extracts were were prepared. prepared. The extracts The were extracts probed with were an anti-phosphotyrosine probed with an antiantibody.-phosphotyrosine As in Figure 3antibody., shown areAs alsoin Figure H3 and 3, αshown-tubulin are blots also toH3 verify and α the-tubulin quality blots of the to subcellular verify the antiantibody.-phosphotyrosine As in Figure 3antibody., shown areAs alsoin Figure H3 and 3, αshown-tubulin are blots also toH3 verify and α the-tubulin quality blots of the to subcellular verify the qualityfractionation, of the subcellular as well as blotting fractionation, of whole as cellwell extracts as blotting (WCE) of whole with antibodiescell extracts for (WCE) NS1 and with NP antibodies to verify qualityfractionation, of the subcellular as well as blotting fractionation, of whole as cellwell extracts as blotting (WCE) of whole with antibodiescell extracts for (WCE) NS1 and with NP antibodies to verify forequal NS1 NS1 and expression NP to verify of NS1equal and NS1 uniform expression infection of NS1 of and cells uniform with the infection different of viruses. cells with the different forequal NS1 NS1 and expression NP to verify of NS1equal and NS1 uniform expression infection of NS1 of and cells uniform with the infection different of viruses. cells with the different viruses. viruses. 4. Discussion 4. Discussion

Acquiring a target motif for an SH3 domain-mediated interaction provides a convenient Acquiring a target motif for an SH3 domain-mediated interaction provides a convenient 4.strategy Discussion for viruses to hijack key signaling pathways that regulate the behavior of their host cells. 4.strategy Discussion for viruses to hijack key signaling pathways that regulate the behavior of their host cells. The high-affinity Crk SH3 binding site in the carboxyterminus of the IAV NS1 protein is an interesting The high-affinity Crk SH3 binding site in the carboxyterminus of the IAV NS1 protein is an interesting Acquiring a target motif for an SH3 domain-mediated interaction provides a convenient strategy Acquiring a target motif for an SH3 domain-mediated interaction provides a convenient strategy example of the ease of such virus-host interaction evolution. As highlighted by the recombinant example of the ease of such virus-host interaction evolution. As highlighted by the recombinant for viruses to hijack key signaling pathways that regulate the behavior of their host cells. The for viruses to hijack key signaling pathways that regulate the behavior of their host cells. The IAV strain A/WSN-NS1WSN(T215P) used in this study, a single nucleotide change in the segment 8 of IAV strain A/WSN-NS1WSN(T215P) used in this study, a single nucleotide change in the segment 8 of high-affinity Crk SH3 binding site in the carboxyterminus of the IAV NS1 protein is an interesting high-affinity Crk SH3 binding site in the carboxyterminus of the IAV NS1 protein is an interesting the viral genome to change an ACT codon into CCT is sufficient to give rise to an NS1 protein with the viral genome to change an ACT codon into CCT is sufficient to give rise to an NS1 protein with example of the ease of such virus-host interaction evolution. As highlighted by the recombinant IAV example of the ease of such virus-host interaction evolution. As highlighted by the recombinant IAV a capacity for fundamentally altering host cell physiology by taking the control of Crk-dependent a capacity for fundamentally altering host cell physiology by taking the control of Crk-dependent strain A/WSN-NS1WSN(T215P) used in this study, a single nucleotide change in the segment 8 of the viral strain A/WSN-NS1WSN(T215P) used in this study, a single nucleotide change in the segment 8 of the viral signaling pathways. The degree of this control can be quite remarkable as evidenced by the dramatic signaling pathways. The degree of this control can be quite remarkable as evidenced by the dramatic genome to change an ACT codon into CCT is sufficient to give rise to an NS1 protein with a capacity genome to change an ACT codon into CCT is sufficient to give rise to an NS1 protein with a capacity relocalization of cellular Crk proteins to the nucleus described in this study. relocalization of cellular Crk proteins to the nucleus described in this study. for fundamentally altering host cell physiology by taking the control of Crk-dependent signaling for fundamentally altering host cell physiology by taking the control of Crk-dependent signaling pathways. The degree of this control can be quite remarkable as evidenced by the dramatic pathways. The degree of this control can be quite remarkable as evidenced by the dramatic relocalization of cellular Crk proteins to the nucleus described in this study. relocalization of cellular Crk proteins to the nucleus described in this study. Despite our present findings as well as other effects on the host cell previously assigned to the Despite our present findings as well as other effects on the host cell previously assigned to the Crk SH3 interaction motif of NS1 [21, 22, 43, 44], the overall role of SH3 binding capacity of NS1 in Crk SH3 interaction motif of NS1 [21, 22, 43, 44], the overall role of SH3 binding capacity of NS1 in supporting IAV replication and pathogenesis remains unclear. Should this property alone provide a supporting IAV replication and pathogenesis remains unclear. Should this property alone provide a clear-cut replicative or immune evasion advantage, it would quickly become fixed in IAV evolution clear-cut replicative or immune evasion advantage, it would quickly become fixed in IAV evolution also in humans, which has not happened. Mutations analogous to our A/WSN-NS1WSN(T215P) mutant also in humans, which has not happened. Mutations analogous to our A/WSN-NS1WSN(T215P) mutant of the human IAV strains A/Udorn/72 and the 2009 Swine Flu pandemic virus (A/California/04/09) of the human IAV strains A/Udorn/72 and the 2009 Swine Flu pandemic virus (A/California/04/09) to introduce an SH3 binding site in NS1 did not result in enhanced viral replication [40, 45]. On the to introduce an SH3 binding site in NS1 did not result in enhanced viral replication [40, 45]. On the other hand, another human IAV strain, A/PR8/8/34, was shown to benefit from the introduction of other hand, another human IAV strain, A/PR8/8/34, was shown to benefit from the introduction of an SH3 binding motif to its NS1 and was more pathogenic in mice [46]. It is tantalizing to note that an SH3 binding motif to its NS1 and was more pathogenic in mice [46]. It is tantalizing to note that

Viruses 2016, 8, 101 11 of 15 Viruses 2016, 8, 101 11 of 15

Despite our present findings as well as other effects on the host cell previously assigned to the Despite our present findings as well as other effects on the host cell previously assigned to the Crk SH3 interaction motif of NS1 [21,22,43,44], the overall role of SH3 binding capacity of NS1 in Crk SH3 interaction motif of NS1 [21,22,43,44], the overall role of SH3 binding capacity of NS1 in supporting IAV replication and pathogenesis remains unclear. Should this property alone provide a supporting IAV replication and pathogenesis remains unclear. Should this property alone provide a clear-cut replicative or immune evasion advantage, it would quickly become fixed in IAV evolution clear-cut replicative or immune evasion advantage, it would quickly become fixed in IAV evolution also in humans, which has not happened. Mutations analogous to our A/WSN-NS1WSN(T215P) mutant also in humans, which has not happened. Mutations analogous to our A/WSN-NS1WSN(T215P) mutant of the human IAV strains A/Udorn/72 and the 2009 Swine Flu pandemic virus (A/California/04/09) of the human IAV strains A/Udorn/72 and the 2009 Swine Flu pandemic virus (A/California/04/09) to introduce an SH3 binding site in NS1 did not result in enhanced viral replication [40,45]. On the to introduce an SH3 binding site in NS1 did not result in enhanced viral replication [40,45]. On the other hand, another human IAV strain, A/PR8/8/34, was shown to benefit from the introduction other hand, another human IAV strain, A/PR8/8/34, was shown to benefit from the introduction of an SH3 binding motif to its NS1 and was more pathogenic in mice [46]. It is tantalizing to note of an SH3 binding motif to its NS1 and was more pathogenic in mice [46]. It is tantalizing to note that the 1918 pandemic Spanish flu virus A/Brevig Mission/1/18/H1N1 is one of the known human that the 1918 pandemic Spanish flu virus A/Brevig Mission/1/18/H1N1 is one of the known human IAV strains that naturally contains this sequence motif, suggesting a positive contribution to viral IAV strains that naturally contains this sequence motif, suggesting a positive contribution to viral fitness in this context [21]. It is likely that the utility of hijacking Crk signaling depends on a complex fitness in this context [21]. It is likely that the utility of hijacking Crk signaling depends on a complex combination of other functional variables of IAV that are not only determined by the sequence variation combination of other functional variables of IAV that are not only determined by the sequence variation in the multifunctional NS1 protein itself, but also encoded by other segments of its genome. in the multifunctional NS1 protein itself, but also encoded by other segments of its genome. In addition to enhancing PI3K activation [21] via re-organization of the PI3K-Crk-complex [22], a In addition to enhancing PI3K activation [21] via re-organization of the PI3K-Crk-complex [22], a functional Crk SH3 binding site of NS1 has previously been linked to suppression of c-Jun N-terminal functional Crk SH3 binding site of NS1 has previously been linked to suppression of c-Jun N-terminal kinase-activating transcriptional factor 2 (JNK-ATF2) pathway [43] and to an inhibition of the tyrosine kinase-activating transcriptional factor 2 (JNK-ATF2) pathway [43] and to an inhibition of the tyrosine kinase c-Abl [44]. Our current data suggest that this list may have to be extended to include many kinase c-Abl [44]. Our current data suggest that this list may have to be extended to include many more of the diverse cellular functions of the Crk protein family. It should mentioned, however, that more of the diverse cellular functions of the Crk protein family. It should mentioned, however, that the interferon-antagonizing effects of NS1 do not fall into this category, and have been shown to be the interferon-antagonizing effects of NS1 do not fall into this category, and have been shown to be independent of Crk SH3 binding [21]. independent of Crk SH3 binding [21]. Nuclear relocalization of the bulk of cellular Crk proteins can be expected to affect several Nuclear relocalization of the bulk of cellular Crk proteins can be expected to affect several cytoplasmic Crk functions. However, phosphorylation and protein interactions are probably more cytoplasmic Crk functions. However, phosphorylation and protein interactions are probably more relevant in regulation of these functions than the total cytoplasmic Crk concentration. This would relevant in regulation of these functions than the total cytoplasmic Crk concentration. This would explain why we did not observe the PI3K-NS1-Crk complex-dependent enhancement of PI3K activity explain why we did not observe the PI3K-NS1-Crk complex-dependent enhancement of PI3K activity in cells expressing an NS1 mutant that was forced to remain cytoplasmic and thus unable to move in cells expressing an NS1 mutant that was forced to remain cytoplasmic and thus unable to move Crk into the nucleus. Perhaps more important than the reduction in the amount of cytoplasmic Crk Crk into the nucleus. Perhaps more important than the reduction in the amount of cytoplasmic Crk may indeed be the triggering of new signaling events in the nucleus induced by NS1-mediated nuclear may indeed be the triggering of new signaling events in the nucleus induced by NS1-mediated nuclear transportation of Crk. transportation of Crk. Previous studies on cancer biology have described triggering of major signaling events and Previous studies on cancer biology have described triggering of major signaling events and outcomes caused by nuclear transport of Crk proteins. CrkII has been reported to participate in outcomes caused by nuclear transport of Crk proteins. CrkII has been reported to participate in apoptosis by activating caspases and binding to the nuclear cell cycle regulator, Wee1 through CrkII apoptosis by activating caspases and binding to the nuclear cell cycle regulator, Wee1 through CrkII SH2 domain [31,32]. On the other hand, CrkL has been reported to bind via its SH2 domain to tyrosine SH2 domain [31,32]. On the other hand, CrkL has been reported to bind via its SH2 domain to tyrosine phosphorylated Stat5 [34,47]. The complex can translocate into the nucleus to bind Stat5-responsive phosphorylated Stat5 [34,47]. The complex can translocate into the nucleus to bind Stat5-responsive elements followed by regulation of gene expression [48,49]. It will be interesting to see how closely elements followed by regulation of gene expression [48,49]. It will be interesting to see how closely NS1-mediated nuclear relocalization of Crk recapitulates these events, and to what extent the complex NS1-mediated nuclear relocalization of Crk recapitulates these events, and to what extent the complex with NS1 redirects Crk to alternative nuclear protein complexes and functions. with NS1 redirects Crk to alternative nuclear protein complexes and functions. As a demonstration that NS1-mediated nuclear transport of Crk proteins can indeed As a demonstration that NS1-mediated nuclear transport of Crk proteins can indeed reprogram nuclear signal transduction pathways, we showed the appearance of a novel nuclear reprogram nuclear signal transduction pathways, we showed the appearance of a novel nuclear tyrosine phosphorylated protein with an estimated molecular weight (MW) of 135 kDa (pp. 135). tyrosine phosphorylated protein with an estimated molecular weight (MW) of 135 kDa (pp. 135). However, despite the established role of Crk proteins in regulating cellular pTyr protein levels, it However, despite the established role of Crk proteins in regulating cellular pTyr protein levels, it should be noted that we cannot exclude the possibility that some SH3-dependent function of NS1 should be noted that we cannot exclude the possibility that some SH3-dependent function of NS1 other than the observed robust nuclear transport of Crk could account for the associated changes in other than the observed robust nuclear transport of Crk could account for the associated changes in nuclear protein tyrosine phosphorylation. nuclear protein tyrosine phosphorylation. The 135 kDa size of the novel pTyr-decorated nuclear protein matches with the major cytoplasmic The 135 kDa size of the novel pTyr-decorated nuclear protein matches with the major cytoplasmic tyrosine-phosphorylated protein partner of Crk proteins, the p130Cas [50,51]. However, while this tyrosine-phosphorylated protein partner of Crk proteins, the p130Cas [50,51]. However, while this remains a possible scenario, so far we have not been able to prove that cytoplasmic p130Cas is remains a possible scenario, so far we have not been able to prove that cytoplasmic p130Cas is transported into the nucleus as a part of an NS1-Crk-p130Cas complex. Another candidate for pp. 135 transported into the nucleus as a part of an NS1-Crk-p130Cas complex. Another candidate for pp. 135 that we have considered, but likewise not been able to prove is c-Abl, a partially nuclear [52] tyrosine that we have considered, but likewise not been able to prove is c-Abl, a partially nuclear [52] tyrosine kinase that can be activated by Crk [ 53] and undergo autophosphorylation [ 54]. However, since Crk kinase that can be activated by Crk [ 53] and undergo autophosphorylation [ 54]. However, since Crk Viruses 2016, 8, 101 12 of 15 Viruses 2016, 8, 101 12 of 15 uses its N-terminal SH3 domain for binding to c-Abl [ 55], this interaction could take place only after uses its N-terminal SH3 domain for binding to c-Abl [ 55], this interaction could take place only after dissociation of the NS1-Crk complex following its nuclear entry. At any case, further studies on the dissociation of the NS1-Crk complex following its nuclear entry. At any case, further studies on the identity of pp135 as well as comprehensive analyses on the changes in the nuclear phosphoproteome identity of pp135 as well as comprehensive analyses on the changes in the nuclear phosphoproteome induced by NS1-mediated nuclear transport of Crk proteins in IAV-infected cells clearly warrants induced by NS1-mediated nuclear transport of Crk proteins in IAV-infected cells clearly warrants further experimental attention in order to better characterize the functional significance of this novel further experimental attention in order to better characterize the functional significance of this novel function of NS1. function of NS1. IAV is an unusual RNA virus in the sense that it replicates in the nucleus of the host cell. Thus, it is IAV is an unusual RNA virus in the sense that it replicates in the nucleus of the host cell. Thus, it is easy to understand why manipulation of the nuclear environment would be relevant for promoting the easy to understand why manipulation of the nuclear environment would be relevant for promoting the IAV life cycle. Ludwig and colleagues have reported that activation of the apoptotic effector caspase-3 IAV life cycle. Ludwig and colleagues have reported that activation of the apoptotic effector caspase-3 at late stages of the IAV replication cycle is required for efficient nuclear exit of viral RNP complexes [56]. at late stages of the IAV replication cycle is required for efficient nuclear exit of viral RNP complexes [56]. Given the previous reports on the capacity of nuclear Crk protein to promote apoptosis [31,32], a role Given the previous reports on the capacity of nuclear Crk protein to promote apoptosis [31,32], a role of NS1-mediated nuclear transport of Crk in facilitating vRNP release from the nucleus poses one of NS1-mediated nuclear transport of Crk in facilitating vRNP release from the nucleus poses one potentially interesting possibility. Since lamins are important caspase substrates and key components potentially interesting possibility. Since lamins are important caspase substrates and key components of the nuclear lamina, we have initiated studies on lamin cleavage and integrity of the nuclear lamina of the nuclear lamina, we have initiated studies on lamin cleavage and integrity of the nuclear lamina during IAV infection. Our preliminary data suggest that viruses expressing Crk binding-competent during IAV infection. Our preliminary data suggest that viruses expressing Crk binding-competent NS1 proteins could indeed induce lamin A/C cleavage and induce more extensive changes in the NS1 proteins could indeed induce lamin A/C cleavage and induce more extensive changes in the nuclear morphology than do viruses with NS1 proteins that lack the SH3 binding motif [57]. nuclear morphology than do viruses with NS1 proteins that lack the SH3 binding motif [57]. The present results further emphasize the role of Crk proteins as host cell interaction partners The present results further emphasize the role of Crk proteins as host cell interaction partners of IAV, although much work remains to be done to characterize the detailed nuclear functions of Crk of IAV, although much work remains to be done to characterize the detailed nuclear functions of Crk protein relocalization to the nucleus by NS1, and the significance of this reprogrammed signaling for protein relocalization to the nucleus by NS1, and the significance of this reprogrammed signaling for IAV replication and pathogenesis. Nevertheless, the remarkable potential of SH3 binding-competent IAV replication and pathogenesis. Nevertheless, the remarkable potential of SH3 binding-competent NS1 proteins to robustly relocate a key family of host cell signaling factors from the cytoplasm to the NS1 proteins to robustly relocate a key family of host cell signaling factors from the cytoplasm to the nucleus attests to the extensive consequences that adopting a short protein interaction motif by a viral nucleus attests to the extensive consequences that adopting a short protein interaction motif by a viral protein can have, and suggests that the role of the NS1-Crk interaction in cell biology of IAV may be protein can have, and suggests that the role of the NS1-Crk interaction in cell biology of IAV may be broader than we have so far appreciated. broader than we have so far appreciated.

Acknowledgments: We are grateful for the Biomedicum Imaging Unit for help in microscopy studies. We thank Acknowledgments: We are grateful for the Biomedicum Imaging Unit for help in microscopy studies. We thank Krister Melén for help constructing the recombinant viruses, Virpi Syvälahti for expert technical assistance, and Krister Melén for help constructing the recombinant viruses, Virpi Syvälahti for expert technical assistance, and the rest of Saksela lab members for help and discussions. This study was supported by grants to K.S. from the rest of Saksela lab members for help and discussions. This study was supported by grants to K.S. from the Academy of Finland, Helsinki University Central Hospital Research Council, Biocentrum Helsinki, and the the Academy of Finland, Helsinki University Central Hospital Research Council, Biocentrum Helsinki, and the Sigrid Juselius Foundation, and by grants to L.Y. from Understödsföreningen Liv och Hälsa, Suomen Tiedesäätiö, Sigrid Juselius Foundation, and by grants to L.Y. from Understödsföreningen Liv och Hälsa, Suomen Tiedesäätiö, and Waldemar von Frenckells stiftelse. L.Y. was supported in part by University of Helsinki Doctoral School in and Waldemar von Frenckells stiftelse. L.Y. was supported in part by University of Helsinki Doctoral School in Health Sciences. Health Sciences. Author Contributions: L.Y., R.F., I.J., and K.S. conceived and designed the experiments; L.Y., I.K., and R.F. Author Contributions: L.Y., R.F., I.J., and K.S. conceived and designed the experiments; L.Y., I.K., and R.F. performed the experiments; L.Y., I.K., R.F., I.J., and K.S. analyzed the data; R.F. and I.J. contributed reagents; L.Y. performed the experiments; L.Y., I.K., R.F., I.J., and K.S. analyzed the data; R.F. and I.J. contributed reagents; L.Y. and K.S. wrote the paper. and K.S. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest.

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