SH3 DOMAIN MEDIATED VIRUS- HOST CELL INTERACTIONS BY THE NONSTRUCTURAL PROTEIN 1 (NS1) OF INFLUENZA A VIRUS LEENA YLÖSMÄKI UNIVERSITY OF HELSINKI FACULTY OF MEDICINE SH3 domain mediated virus-host cell interactions by the nonstructural protein 1 (NS1) of influenza A virus Department of Virology Faculty of Medicine University of Helsinki Finland Leena Ylösmäki ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Medicine, University of Helsinki, for public examination in the Lecture Hall 2, Haartmaninkatu 3, on June 28th, 2016, at 12 noon. Helsinki 2016 SUPERVISED BY: Kalle Saksela, MD, PhD, Professor Department of Virology Faculty of Medicine University of Helsinki Finland REVIEWED BY: Tero Ahola, PhD, Docent Department of Food and Environmental Sciences Faculty of Agriculture and Forestry University of Helsinki Finland Denis Kainov, PhD, Docent Institute for Molecular Medicine Finland (FIMM) University of Helsinki Finland OPPONENT: Varpu Marjomäki, PhD, Docent Department of Biological and Environmental Science Faculty of Mathematics and Science University of Jyväskylä Finland ISBN 978-951-51-2283-4 (paperback) ISBN 978-951-51-2284-1 (PDF) Picaset Oy http://ethesis.helsinki.fi Helsinki 2016 2 To my family 3 Table of Contents LIST OF ORIGINAL PUBLICATIONS .................................................................................. 7 ABSTRACT ..................................................................................................................... 8 ABBREVIATIONS ............................................................................................................ 9 1 REVIEW OF LITERATURE ....................................................................................... 11 1.1 Influenza A virus ............................................................................................... 11 1.1.1 Disease and epidemics ............................................................................... 11 1.1.2 Structure of the virion ................................................................................ 12 1.2 Influenza A virus life cycle ................................................................................ 13 1.2.1 Entry .......................................................................................................... 13 1.2.2 Nuclear import 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 1.2.5 Virion assembly and budding ..................................................................... 16 1.3 Influenza A virus NS1 protein ........................................................................... 16 1.3.1 Stucture of NS1 .......................................................................................... 16 1.3.2 Intracellular localization of NS1 .................................................................. 17 1.3.3 Post-translational modifications of NS1 ...................................................... 17 1.3.4 Inhibition of interferon production by NS1 ................................................ 18 1.3.4.1 Inhibition of the RIG-I pathway .......................................................... 19 1.3.4.2 Inhibition of host gene expression ..................................................... 20 1.3.4.3 JNK pathway ...................................................................................... 20 1.3.4.4 Inhibition of the activity of antiviral proteins PKR and OAS................. 21 1.3.5 NS1 in apoptotic pathways ......................................................................... 22 1.3.6 Interactions of NS1 with other host cell factors .......................................... 22 1.3.6.1 PDZ domain mediated interactions .................................................... 22 1.3.6.2 SH3 domain mediated interactions .................................................... 23 1.3.7 Regulation of viral RNA and protein synthesis by NS1 ................................ 23 1.4 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 1.4.3 Induction of the PI3K/Akt pathway by NS1 protein..................................... 26 1.5 SH3 domains .................................................................................................... 27 1.5.1 SH3 domain structure ................................................................................ 28 1.5.2 SH3 domain ligand binding motifs .............................................................. 28 4 1.5.2.1 Typical ligand binding motifs .............................................................. 28 1.5.2.2 Atypical ligand binding motifs ............................................................ 29 1.5.3 Affinity and specificity of SH3 domains ....................................................... 29 1.5.4 Viral proteins as SH3 domain ligands .......................................................... 30 1.6 Crk adaptor proteins ........................................................................................ 31 1.6.1 Stucture and binding specificities of Crk proteins ....................................... 32 1.6.2 Regulation of Crk proteins .......................................................................... 33 1.6.3 Nuclear import and export of Crk proteins ................................................. 34 1.6.4 Biological function of Crk proteins .............................................................. 34 1.6.4.1 Crk in PI3K signaling ........................................................................... 35 1.6.4.2 Crk in apoptotic pathways .................................................................. 36 2 AIMS OF THE STUDY ............................................................................................. 37 3 MATERIALS AND METHODS .................................................................................. 38 3.1 Cell culture and transfections (I-III) ................................................................... 38 3.2 Plasmid constructs ........................................................................................... 38 3.2.1 Eukaryotic expression vectors (I-III) ............................................................ 38 3.2.2 Bacterial expression vectors (I,II) ................................................................ 39 3.3 Viruses and virus infections (I,III) ...................................................................... 39 3.4 Antibodies (I-III)................................................................................................ 40 3.5 Protein sequence alignment (I)......................................................................... 40 3.6 Recombinant protein production, purification and phage screening (I,II) ......... 41 3.7 Protein precipitation and Western blot (I-III) .................................................... 41 3.8 Detection of phosphorylated proteins (I-III)...................................................... 41 3.9 Dual-luciferase assays (I) .................................................................................. 41 3.10 In vitro protein binding assays (I,II) ................................................................. 41 3.11 Cell fractionation (III) ...................................................................................... 42 3.12 Immunostaining and imaging (III) ................................................................... 42 4 RESULTS ............................................................................................................... 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 4.1.2 Functional role for the Crk-NS1 interaction (I,II) ......................................... 44 4.1.3 Trimeric complex formation upon Crk-NS1 interaction (II) ......................... 45 4.1.4 Functional consequences of the trimeric complex (II)................................. 47 4.2 Localization of the Crk-NS1 complex (III) .......................................................... 47 4.2.1 NS1 mediates nuclear translocation of Crk proteins (III) ............................. 47 5 4.2.2 Functional role of Crk translocation into the nucleus by NS1 (III) ................ 47 5 DISCUSSION .......................................................................................................... 49 5.1 Crk-NS1 interaction and PI3K activation ........................................................... 49 5.2 Crk-NS1-PI3K complex formation ..................................................................... 50 5.3 Nuclear localization of Crk-NS1 complex .......................................................... 51 5.4 Multiple functions of Crk-NS1 interaction......................................................... 52 5.5 Diversity in the SH3 binding motif of NS1 ......................................................... 53 5.6 Significance of the NS1 SH3 binding motif for IAV ............................................ 53 6 CONCLUSIONS .....................................................................................................