The murine cytomegalovirus gp40/m152 inhibits activation of NK cell receptor NKG2D by intracellular retention and cell surface masking of RAE-1g ligand by

Natalia Lis

a Thesis submitted in partial fulfilment of the requirements for the degree of

Doctor of Philosophy in Cell Biology

Approved Dissertation Committee ______Prof. Dr. Sebastian Springer Jacobs University Bremen

Prof. Dr. Susanne Illenberger Jacobs University Bremen

Prof. Dr. Wolfram Brune Heinrich-Pette-Institut Hamburg

Date of Defense: 04.09.2020 Life Sciences & Chemistry

2 Statutory Declaration

Family Name, Given/First Name Natalia Lis

Matriculation number 20331750

What kind of thesis are you submitting: PhD thesis Bachelor-, Master- or PhD-Thesis

English: Declaration of Authorship I hereby declare that the thesis submitted was created and written solely by myself without any external support. Any sources, direct or indirect, are marked as such. I am aware of the fact that the contents of the thesis in digital form may be revised with regard to usage of unauthorized aid as well as whether the whole or parts of it may be identified as plagiarism. I do agree my work to be entered into a database for it to be compared with existing sources, where it will remain in order to enable further comparisons with future theses. This does not grant any rights of reproduction and usage, however. The Thesis has been written independently and has not been submitted at any other university for the conferral of a PhD degree; neither has the thesis been previously published in full. German: Erklärung der Autorenschaft (Urheberschaft) Ich erkläre hiermit, dass die vorliegende Arbeit ohne fremde Hilfe ausschließlich von mir erstellt und geschrieben worden ist. Jedwede verwendeten Quellen, direkter oder indirekter Art, sind als solche kenntlich gemacht worden. Mir ist die Tatsache bewusst, dass der Inhalt der Thesis in digitaler Form geprüft werden kann im Hinblick darauf, ob es sich ganz oder in Teilen um ein Plagiat handelt. Ich bin damit einverstanden, dass meine Arbeit in einer Datenbank eingegeben werden kann, um mit bereits bestehenden Quellen verglichen zu werden und dort auch verbleibt, um mit zukünftigen Arbeiten verglichen werden zu können. Dies berechtigt jedoch nicht zur Verwendung oder Vervielfältigung. Diese Arbeit wurde in der vorliegenden Form weder einer anderen Prüfungsbehörde vorgelegt noch wurde das Gesamtdokument bisher veröffentlicht.

Date, Signature

3

This work was funded by Deutsche Forschungsgemeinschaft (SP583/11-1) and by the Tönjes Vagt Foundation of Bremen (XXXII) to Sebastian Springer.

4

Part of this thesis is submitted to JCS peer-reviewed journal

5 Acknowledgments

First of all, I would like to express my gratitude to Prof. Sebastian Springer for giving me an opportunity to join your group and work on this exciting and challenging project. You gave me a perfect amount of supervision, guidance, and space to grow professionally.

I would like to thank my thesis committee members, Prof. Susanne Illenberger and Prof. Wolfram Brune, for your dedication, time, and effort while reviewing my research work.

I would like to thank Springer group current and former members: Swapnil Ghanwat, Ankur Saikia, Raghavendra Anjappa, Britta Borchert, Cindy Dirscherl, Esam Abualrous, Sebastián Montealegre, and Sujit Verma for a great working environment, and especially Ursula Wellbrock for being the most trusted, patient and helpful colleague ever.

Special thanks go to Venkat Raman Ramnarayan and Zeynep Hein for teaching me all the methods and scientific approaches necessary to succeed with my work and become a scientist, and endless discussions of my work.

Many thanks go to my students, Bersal Williams and Miriam Herbert, for your great help with the project and the chance to improve my teaching skills.

The project would not develop so well without much valuable scientific advice from excellent scientists like Dr. Linda Janssen, Dr. Malgorzata Garstka, Dr. Susanne Fritzsche, Dr. Anne Halenius, Dr. Benedict Chambers, Prof. Martin Messerle, and Prof. Hartmut Hengel. Thank you for all your input.

I would like to thank my friends in Bremen for their company and all the fun we had during the past five years. I enjoyed each of our meetings and celebrations together. You made this time very joyful and precious.

This time would be extremely difficult without the support and encouragement from my awesome mum Jola and my super-smart sister Paulina. Dziękuję!

Finally, I would like to thank Pawel for being a supportive, patient, and wonderful partner throughout this time.

6 Abstract

NKG2D is a crucial Natural Killer (NK) cell activating receptor, and the murine cytomegalovirus (MCMV) employs multiple immunoevasins in order to avoid

NKG2D-mediated activation. One of the MCMV immunoevasins, gp40 (m152), downregulates the cell surface NKG2D ligand, RAE-1g, thus limiting NK cell activation. My study establishes the molecular mechanism by which gp40 retains

RAE-1g in the secretory pathway. Using flow cytometry and pulse chase analysis,

I demonstrate that gp40 retains RAE-1g in the early secretory pathway, and that this effect depends on the binding of gp40 to a host , TMED10, a member of the p24 protein family. I also show that the TMED10-based retention mechanism can be saturated, and that gp40 has a backup mechanism as it masks RAE-1g on the cell surface, blocking the interaction with the NKG2D receptor and thus NK cell activation.

7 List of abbreviations

ADAM - A disintegrin and metalloprotease ADCC - Antibody-dependent cellular cytotoxicity AUF1 - AU-rich element RNA-binding protein 1 BiP - Binding immunoglobulin protein cGAMP - Cyclic guanosine monophosphate–adenosine monophosphate cGAS - Cyclic GMP-AMP synthase, cGAMP synthase CK2 - Casein kinase 2 CLP - Common lymphoid progenitors COP - Coat protein complex DAF - Decay-accelerating factor DAMP - Damage-associated molecular pattern DAP - Disulphide adaptor molecule EGFR - Epidermal growth factor receptor ERGIC - ER-Golgi intermediate compartment FasL - Fas ligand GM-CSF - Granulocyte–macrophage colony-stimulating factor GOLD - Golgi dynamic (domain) gp40LM - gp40 linker mutant gp40WT - gp40 wild type GPCR - G-protein-coupled receptors GPI - Glycosylphosphatidylinositol (anchor) H60 - Histocompatibility antigen 60 HCMV - cytomegalovirus HDAC3 - Histone deacetylase 3 hpi - Hours post IE - Immediate early IL - Interleukin ILC - Innate lymphoid cells IMP3 - Insulin-like growth factor 2 mRNA-binding protein 3 INF - Interferon IRF3 - Interferon regulatory factor 3 ITAM - Immunoreceptor tyrosine-based activation motif KIR - Killer-cell immunoglobulin-like receptors Klrk1 - Killer Cell Lectin Like Receptor K1 () LAMP-1 - Lysosomal-Associated Membrane Protein-1 LPS - Lipopolisacharide MCMV - Mouse cytomegalovirus metastamiR, miR - Metastasis-associated miRNA MICA/B - MHC class I polypeptide–related sequences A/B

8 MMP - Matrix metalloproteinase MULT-1 - Murine UL16-binding protein-like transcript-1 NCAM - Neural cell adhesion molecule ? NF-kB - Nuclear Factor kappa-light-chain-enhancer of activated B cells NK cell - Natural killer (cell) NKC - NK gene complex NKG2D-L - Long NKG2DR isoform NKG2D-S - Short NKG2DR isoform NKG2DL - NKG2D ligands NKG2DR - NKG2D receptor ORF - Open reading frames PAMP - Pathogen-associated molecular pattern PI3K - Phosphatidylinositol 3-kinase PLC - Peptide loading complex RAE-1 - Retinoic acid early-inducible protein 1 RBP - RNA binding protein SLT - Secondary lymphoid tissues SNP - Single nucleotide polymorphism SPR - Surface plasmon resonance STING - Stimulator of interferon genes STR - Short tandem repeat SV - Sedimentation velocity ultracentrifugation TBK1 - TANK Binding Kinase 1 TCR - receptor TLR - Toll like receptor TNF - Tumor necrosis factor TRAIL - TNF-related apoptosis-inducing ligand UL - Unique long (region of the HCMV genome) ULBP - UL16-binding protein US - Unique short (region of the HCMV genome) VAC - Viral assembly compartment vICA - Viral Inhibitor of Caspase-8 Activation YINM - Tyrosine-based motif

9 List of figures

Figure 1-1. Cytomegalovirus genome organization ...... 19

Figure 1-2. RAE-1g amino acid sequence...... 36

Figure 1-3. Overview of human and mouse immunoevasins that target NKG2D ligands...... 40

Figure 1-4. Amino acid sequence of gp40...... 46

Figure 1-5. Amino acid sequence alignment of RAE-1 isoforms...... 51

Figure 1-6. Crystal structures of NKG2D ligands, NKG2D receptor, HCMV and MCMV immunoevasins, and mouse MHC class I H-2Kb...... 53

Figure 1-7. Enrichment of p24 at the cell surface and in the whole cell lysate during HCMV infection (176)...... 61

Figure 3-1. Mouse and human NKG2D ligands...... 89

Figure 3-2 . gp40 retains murine MHC class I proteins in the early secretory pathway...... 92

Figure 3-3. Intracellular retention of class I depends on the binding of the gp40 linker to TMED10...... 92

Figure 3-4. Preliminary data: gp40 downregulates cell surface levels of RAE-1g in murine and human cells...... 94

Figure 3-5. Preliminary data: Cell surface gp40 masks RAE-1g from CX1 recognition...... 96

Figure 3-6. gp40 might block the interaction between RAE-1g and NKG2D. .... 97

Figure 3-7. Schematic summary of the two putative modes of action of gp40 on RAE-1g...... 98

Figure 3-8. Work program...... 102

Figure 5-1. Crystal structure of gp40 and RAE-1g...... 119

Figure 5-2. RAE-1g and class I expression in cells co-expressing gp40 mutants ...... 120

Figure 5-3. Glycosylation of gp40 E28A/D113A/D236A...... 121

10 Figure 5-4. Oligomerization of gp40...... 123

Figure 5-5. Glycosylation of RAE-1g...... 127

Figure 5-6. Digest of RAE-1g using different treatment conditions...... 128

Figure 5-7. RAE-1g trafficking in TMED10-deletion cells...... 129

Figure 5-8. Quantification of RAE-1g trafficking in TMED10-deletion cells. ... 130

Figure 5-9. Morphology of MCMV-infected cells...... 132

Figure 5-10. gp40 intracellular expression and cell surface expression...... 133

Figure 5-11. IE1 intracellular expression...... 134

Figure 5-12. H-2Kb cell surface expression...... 135

Figure 5-13. RAE-1g cell surface expression...... 136

11 List of tables

Table 1-1. Taxonomy and nomenclature of cytomegaloviruses (9)...... 17

Table 1-2. Human NK cell activating and inhibiting receptors (45–48) ...... 23

Table 1-3. NKG2D receptor isoforms (57)...... 28

Table 1-4. Human and mouse NKG2D ligands (65-69)...... 29

Table 1-5. NKG2D ligand RNA expression in different human tissues (89)...... 33

Table 1-6. Affinity of RAE-1 isoforms to the NKG2D receptor and to gp40...... 49

Table 1-7. Nomenclature of p24 proteins in mammals (162)...... 56

Table 1-8. RNA expression of some p24 proteins in different human tissues (89) . 56

Table 2-1. Primers used for cloning of gp40 mutants and myc-tagged gp40 ...... 64

Table 2-2. Primers used for cloning of RAE-1g mutants ...... 65

Table 2-3. Antibody dilutions used for cell staining and flow cytometry ...... 67

Table 3-1. Work schedule of the project ...... 113

Table 3-2. Costs for consumables (Euros p.a.) ...... 113

Table 3-3. Total costs of the project ...... 114

Table 5-1. Residues of gp40 and RAE-1g crucial for the formation of a strong complex...... 118

12 Table of contents

1 Introduction ...... 16 1.1 Herpesviruses and cytomegalovirus ...... 16 1.1.1 ...... 16 1.1.2 Morphology ...... 16 1.1.3 Cytomegalovirus ...... 17 1.1.4 Model to study HCMV ...... 17 1.1.5 HCMV life cycle (simplified) ...... 18 1.1.6 HCMV and MCMV genome ...... 18 1.1.7 Viral assembly compartment ...... 19 1.1.8 HCMV disease ...... 20 1.2 Natural killer cells ...... 21 1.2.1 NK cell localization ...... 21 1.2.2 NK cell subsets ...... 21 1.2.3 NK cell function and activation ...... 22 1.2.4 NK cell killing kinetics ...... 26 1.3 NKG2D receptor ...... 26 1.3.1 NKG2D receptor structure ...... 26 1.3.2 NKG2D receptor expression ...... 27 1.3.3 NKG2D receptor isoforms and function ...... 27 1.4 NKG2D ligands ...... 28 1.4.1 NKG2D ligand families ...... 28 1.4.2 NKG2D ligand structure ...... 29 1.4.3 NKG2D genes ...... 30 1.4.4 NKG2D ligand-receptor binding ...... 30 1.4.5 NKG2D ligand expression ...... 31 1.4.6 NKG2D ligands diversity and expression patterns ...... 33 1.5 RAE-1 ...... 35 1.5.1 Expression, structure and function ...... 35 1.5.2 GPI anchor ...... 36 1.5.3 Regulation of RAE-1 expression ...... 37 1.5.4 NKG2DR/RAE-1 structure ...... 38 1.6 Cytomegalovirus immunoevasins ...... 38 1.6.1 Human cytomegalovirus immune evasion of NKG2D ligand-receptor pathway ...... 41 1.6.2 Mouse cytomegalovirus immune evasion of NKG2D ligand-receptor pathway ...... 44 1.7 gp40 ...... 46 1.7.1 gp40 interaction with MHC class I ...... 47 1.7.2 gp40 interaction with RAE-1 ...... 48 1.7.3 gp40 interaction with STING ...... 51

13 1.8 Models to study viral immune evasion ...... 54 1.9 The p24 proteins ...... 55 1.9.1 Nomenclature ...... 55 1.9.2 Expression ...... 56 1.9.3 Structure ...... 57 1.9.4 Cellular localization ...... 58 1.9.5 Function ...... 58 1.9.6 TMED10 and other p24 proteins that interact with gp40 ...... 58 1.10 Open questions and aims of the project ...... 61

2 Materials and methods ...... 64 2.1 Materials ...... 64 2.1.1 Constructs ...... 64 2.2 Methods ...... 66 2.2.1 Cell culture, NK cell isolation, cytotoxicity assays ...... 66 2.2.2 Lentiviral transduction ...... 66 2.2.3 Immunofluorescence microscopy ...... 66 2.2.4 Radioactive labeling and immunoprecipitation ...... 66 2.2.5 Co-immunoprecipitation and re-immunoprecipitation, EndoF1 digest, PNGase digest, SDS-PAGE ...... 67 2.2.6 Immunostaining and flow cytometry ...... 67 2.2.7 MCMV infection ...... 67 2.3 Standard operating procedures ...... 68 2.3.1 SOP: Flow cytometry of mammalian cells ...... 68 2.3.2 SOP: Production of lentiviruses ...... 76 2.3.3 SOP: Lentiviral transduction of cells ...... 81

3 Grant application ...... 88 3.1 State of the art and preliminary work ...... 88 3.1.1 Scientific background of the project ...... 88 3.1.2 Preliminary data ...... 91 3.1.3 Project-related publications ...... 99 3.2 Objectives and work program ...... 99 3.2.1 Anticipated total duration of the project ...... 100 3.2.2 Objectives ...... 100 3.2.3 Work program including proposed research methods ...... 101 3.2.4 Data handling ...... 109 3.2.5 Other information: Not applicable...... 109 3.2.6 Description of proposed investigations involving experiments on , human materials, or animals as well as dual use research of concern: Not applicable...... 109 3.2.7 Information on scientific and financial involvement of international cooperation partners: Not applicable...... 109

14 3.3 Bibliography ...... 109 3.4 Requested modules/funds ...... 112 3.4.1 Basic Module ...... 112 3.4.2 Funding for staff ...... 112 3.4.3 Total costs ...... 114 3.5 Project requirements ...... 114 3.5.1 Employment status information ...... 114 3.5.2 First-time proposal data: Not applicable...... 114 3.5.3 Composition of the project group ...... 114 3.5.4 Cooperation with other researchers ...... 114 3.5.5 Scientific equipment ...... 115 3.5.6 Project-relevant cooperation with commercial enterprises: None...... 115 3.5.7 Project-relevant participation in commercial enterprises: None...... 115 3.5.8 Additional information ...... 115

4 Manuscript ...... 116

5 Additional projects and experiments ...... 118 5.1 gp40 mutant that does not bind to RAE1g ...... 118 5.2 gp40 oligomerization ...... 121 5.3 RAE-1g membrane anchorage ...... 123 5.4 RAE-1g glycosylation ...... 126 5.5 RAE-1g trafficking in TMED10-deletion cells ...... 129 5.6 MCMV infection of mouse cells ...... 131 5.6.1 Intracellular gp40 ...... 132 5.6.2 Intracellular IE1 ...... 133 5.6.3 Cell surface H-2Kb ...... 134 5.6.4 Cell surface RAE-1g ...... 135

6 Outlook ...... 137

7 List of journal publications, conference participations and awards ...... 139

8 References ...... 140

15 1. Introduction

1 Introduction

1.1 Herpesviruses and cytomegalovirus

1.1.1 Herpesviridae The Herpesviridae, or herpesviruses, are a large family of divided into three subfamilies named Alpha-, Beta- and Gammaherpesviruses, that share common morphology, but lack genomic similarities. While Alphaherpesviruses are able to infect a wide variety of species, Beta- and Gammaherpesviruses are more species-restricted (1). The best-studied human herpesviruses (HHVs) are herpes simplex 1 (HSV-1) and HSV-2, (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and Kaposi’s sarcoma-associated herpesvirus (KSHV) (2). Infection symptoms are usually not severe unless the infection occurs in a very young or immunosuppressed individual. Herpesviruses are highly adapted to their hosts and they developed various strategies to modulate the host immune response and cellular machinery. They developed 180 to 220 million years ago, diverged into subfamilies 80 to 60 million years ago, and co-evolution of the herpesviruses and hosts is manifested by the diversity of the immune evasion strategies and corresponding immune responses [1.6]1 (3). They can establish a life-long latency in various cell types such as neurons (Alphaherpesviruses), myeloid cells lineages (Betaherpesviruses), and lymphocytes (Gammaherpesviruses) (1).

1.1.2 Morphology All herpesviruses share common morphology characteristics. The spherical virion consists out of three types of structures: first, an icosahedral shaped capsid containing the tightly packed genome; second, the tegument protein layer that with various proteins important for viral gene transcription and expression, modulation of the host cell metabolism, and host cell antiviral responses; and third, the lipid envelope with different surface glycoproteins crucial for viral entry and infectivity (i.e., ability to establish an infection). On average, the capsid size is about 125 nm, and the diameter of the whole viral particle including the envelope is about 200 nm. The relatively big genomes of herpesviruses range between 125 and 240 kbp (1).

1 Numbers in square brackets are references to other sections of this thesis

16 1. Introduction

1.1.3 Cytomegalovirus Cytomegaloviruses belong to the Betaherpesvirus subfamily. HCMV transmission occurs via contact with urine or saliva, and it can infect a wide range of human tissues, predominantly epithelial cells of glands and mucosal tissues, connective tissues, gastrointestinal tract smooth muscle cells, and vascular endothelial cells. HCMV can replicate in almost any organ, but the highest viral titers are usually present in the liver, lungs, gastrointestinal tract, and retina (4).

1.1.4 Model to study HCMV HCMV infection is strictly restricted to the human host and thus cannot be studied in other animals. Therefore, an comparable animal model is crucial to learn about the mechanisms of the HCMV infection and address questions that cannot be studied in clinical research. Several genera of cytomegaloviruses have been identified, including monkeys and rodent viruses (Table 1-1). One of the best studied and easily accessible models is a mouse cytomegalovirus (MCMV), also known as murid betaherpesvirus 1 (MuHV-1). MCMV and HCMV share a lot of characteristics. For example, they both express immunoevasins, i.e., proteins that inhibit host immune responses, and hence, discoveries made with MCMV allow researchers to predict viral immune evasion strategies of HCMV. A huge advantage of MCMV is that discoveries made in vitro or in the cell culture setting can be further tested in vivo. This allows researchers to study individual viral proteins in an isolated setting, and then test their roles during an infection in the host (5–8).

Table 1-1. Taxonomy and nomenclature of cytomegaloviruses (9).

Formal name Abbreviation Common name

Subfamily Betaherpesvirinae Genus Cytomegalovirus Cercopithecine herpesvirus 5 CeHV-5, SCMV African green monkey cytomegalovirus Cercopithecine herpesvirus 8 CeHV-8, RhCMV Rhesus monkey cytomegalovirus Human herpesvirus 5 HHV-5, HCMV Human cytomegalovirus Pongine herpesvirus 4 PoHV-4, CCMV Chimpanzee cytomegalovirus Genus Muromegalovirus Murid herpesvirus 1 MuHV-1, MCMV Mouse cytomegalovirus Murid herpesvirus 2 MuHV-2 Rat cytomegalovirus

17 1. Introduction

1.1.5 HCMV life cycle (simplified) HCMV enters the host cell either by direct fusion or by . Viral glycoproteins essential for the cell tropism and entry process are gB and gH/gL, and depending on the cell type, additional protein complexes are necessary (10). Next, the capsid is released into the and translocated via the host microtubule system into the nucleus where the viral DNA is released. Expression of viral genes initiates the viral genome replication via the “rolling circle” mechanism, and immune evasion. The newly synthesized viral DNA undergoes initial encapsulation and tegumentation and is then translocated to the cytoplasm. The process is regulated by a nuclear egress complex consisting of viral and host proteins (11). After reaching the cytoplasm, viral particles are re-located to the viral assembly compartment [1.1.7], and they undergo secondary envelopment. The fully formed viruses are finally released at the plasma membrane (12, 13).

1.1.6 HCMV and MCMV genome The HCMV genome is 235 kbp long, and it is the biggest known genome among human viruses. Similarly to other Betaherpesviruses, the genome consists of a linear double-stranded DNA molecule, with an unpaired base on each end. These unpaired bases facilitate circularization when the genome is packed (14).

The genome of HCMV consists of two unique regions called unique long (UL) and unique short (US), which are flanked by internal and terminal inverted repeats

(IRL, IRS, TRL, TRS,). The UL and US regions can individually change their orientation, resulting in four possible versions of the genome (genomic isomers) that are found during infection in equimolar proportion (Figure 1-1). The names of genes that are encoded in the UL and US regions are prefixed with the letters UL or US, respectively (15, 16).

Out of 162 HCMV open reading frames (ORFs), 45 ORFs are essential for replication in fibroblasts, and out of those, 78% are conserved among herpesviruses. 117 ORFs regulate other viral functions such as cell tropism, immune evasion, or expression of surface glycoprotein receptors. Importantly, some genes are negative regulators of replication. It has been proposed that in order to prevent massive infection that would overburden the host, these genes regulate replication specifically in different types of tissues or during latency. For many HCMV ORFs, the function has not been yet

18 1. Introduction discovered. Besides protein coding genes, HCMV genome also encodes polyadenylated non-coding RNAs and regulatory microRNAs (17).

The MCMV genome is about 230 kbp long, and it consists out of a single unique sequence flanked by terminal repeats (TR) at both ends, and to some extent within the genome (Figure 1-1) (18).

Figure 1-1. Cytomegalovirus genome organization

HCMV and MCMV genes are grouped into clusters/families of homologous ORFs, and genes that are orthologous between these two viruses are located in the central parts of their genomes. MCMV genes that are conserved between HCMV and MCMV are designated with an uppercase letter, for example capsid component protein M86 (in HCMV UL86), whereas those lacking sequence similarity with HCMV genes are designated with a lowercase letter, for example MCMV m152 that encodes membrane glycoprotein gp40 [1.7] (15, 17, 18).

With respect to their time of expression, the CMV genes are grouped into early and late expression genes. The first group, referred to as immediate early genes (IE), regulates the early phase of infection, mainly the viral genome replication. Expression of late genes includes production of structural proteins and proteins necessary for the assembly of the new viral particles. Genes encoding immunoevasins belong to both groups (15, 17).

1.1.7 Viral assembly compartment The viral assembly compartment (VAC) is an enlarged structure formed close to the nucleus of infected cells. HCMV modifies existing cellular membranes and mechanisms to create the VAC, which is composed of secretory pathway membranes including membranes from the ER, the ER-Golgi intermediate compartment (ERGIC), the Golgi network, and early endosomes. In infected cells, the nucleus has an altered shape and increased size, and the nuclear membranes are reorganized into the

19 1. Introduction kidney-like shape surrounding the VAC allowing the egress of nucleocapsids to the cytoplasm (19, 20). The microtubule organizing centre supports the structure and microtubule-associated motors sustain the transport of viral capsids (21). The VAC was proposed to have a cylindrical shape or cylindrical layers and it contains multiple host proteins including SNARE family members (endocytic/lysosomal markers), the ER chaperone BiP (immunoglobulin Binding Protein), ESCORT (endosomal sorting complex required for transport) proteins that control cargo incorporation, and HCMV-encoded Fc receptor-like proteins (12, 19, 20, 22, 23).

1.1.8 HCMV disease HCMV infection is common, and it rarely causes serious problems in healthy individuals. The primary infection, although usually asymptomatic, may cause flu-like symptoms including tiredness, fever, and swollen glands. After the primary infection, the virus can establish a life-long latency with low or zero level of detectable virus replication. Latent HCMV persists in many nucleated cells, mainly in CD14+ peripheral blood mononuclear cells, and bone marrow CD34+ and CD33+ cells (24, 25).

CMV transmission occurs through exposure to saliva, tears, urine, stool, breast milk, or semen, as well as via organ and tissue transplantation or blood transfusions. It can also be transmitted from mother to a child during pregnancy, resulting in congenital CMV infection (25, 26).

One group that is especially sensitive to CMV infection are immunocompromised patients, and among those, it can cause life-threatening complications. CMV infection decreases the survival rate among solid organ transplant and stem cell transplant patients and HIV-infected persons. These individuals may develop serious CMV disease resulting in multiple organ inflammation including pneumonitis, retinitis, hepatitis, myocarditis, pancreatitis, or gastroenteritis (27). CMV is also particularly harmful to newborns and young children. Congenital CMV infection can result in vision damage and neurodevelopmental delay or disability, and it is a leading pathogen-related cause of childhood hearing loss worldwide (26, 28).

The CMV seroprevalence rate depends on age, sex, and geographic location, and it is particularly high in communities with a low socioeconomic status. An estimated global mean seroprevalence for the general population varies between 78-88%, with the highest rate in the Eastern Mediterranean region (88-95%) and the lowest rate in

20 1. Introduction

Europe (56-74%) (29).

1.2 Natural killer cells Natural killer (NK) cells were discovered in the 1970s, and they were initially characterized as innate immune cells. Next to B cells or T cells, NK cells are one of the largest group of lymphocytes. The hallmark of NK cells is their ability to kill virally infected and malignantly transformed cells without a prior exposure to the antigen. They are commonly classified as a subset of innate lymphoid cells (ILCs), a group of cells derived from lymphoid progenitor cells in the bone marrow that lack specific antigen receptors arising as a result of genetic rearrangement (30).

1.2.1 NK cell localization The majority of human and mouse NK cells are found in the blood and in organs such as bone marrow, lung, peripheral blood, and liver, and in secondary lymphoid tissues (SLT) including the tonsils, spleen, and lymph nodes. Subsets of NK cells have also been detected in other organs of human and mouse, such as the intestine, skin, bladder, uterus, kidney, pancreas, adipose tissue, and brain (only mouse). All NK cells in human and mouse originate from multipotent hematopoietic stem cells that further progress into common lymphoid progenitors (CLPs). In a multistep process, CLPs acquire or lose expression of various specific cell surface markers before they become fully mature NK cells. The developmental stages of NK cells, even though studied thoroughly, are still not fully understood. The NK cell development in mouse is restricted to the bone marrow, but in humans, it also takes a place in the SLT, and to some extent in other tissues (31–33).

1.2.2 NK cell subsets The most common way to categorize human NK cells in based on the cell surface expression of CD56 antigen. CD56, also called neural cell adhesion molecule (NCAM), belongs to the immunoglobulin superfamily, and it is phenotypic marker of NK cells. However, it is also found on other lymphoid cells, for example gamma delta (γδ) T cells and activated CD8+ T cells, or dendritic cells (34).

The first group, CD56bright NK cells, expresses high levels of CD94 (a family of C-type lectin surface receptors) and low or absent levels of KIRs (Killer-cell immunoglobulin- like receptors) as well as CD16 (also known as FcγRIII). The second group, CD56dim

21 1. Introduction

NK cells, express low levels of CD94 and high levels of KIR and CD16. CD56bright NK cells predominate in the lymph nodes and tonsils, they exhibit better proliferation abilities, rapidly produce cytokines and chemokines in response to target cells, and have a low cytotoxic activity. CD56dim cells predominate in the peripheral blood, they express high levels of perforins, and consequently they are highly cytotoxic against tumours and virally infected cells. It is believed that CD56dim cells arise, at least partially, from CD56bright cells in a series of intermediate developmental stages during which the ratio of CD94, KIR, and CD16 molecules is changing (31, 35).

Mouse NK cell development stages are also defined by expression of distinct cell surface receptors involved in NK cell function, namely the C-type lectin receptors NKG2D, NKG2A, and Ly49; functional markers CD122, DNAM-1, NK1.1 and NCR1; cell adhesion molecules like L-selectin and Leukosialin, as well as integrins CD51 and CD49b. The key NK cell surface markers common between human and mouse are the C-type lectin receptors NKG2A and NKG2D (36).

The relationship between different NK cell subsets, as well as signals that drive the differentiation, are still to be clarified. Some of the transcription factors involved in the NK cell development in human and mouse are T-bet and Eomesodermin (36).

1.2.3 NK cell function and activation The main function of NK cells to recognize and kill virally infected or tumor host cells. They also play a regulatory role by secreting molecules such as interferon gamma (INF-g) or tumor necrosis factor (TNF), that influence dendritic cells, T cells, B cells, and macrophages (37).

NK cells distinguish healthy and sick cells via multiple surface activating and inhibiting receptors. The ratio between the two antagonistic types of signals determines whether an NK cell will be activated (37).

1.2.3.1 NK cell activation There are numerous interactions important for NK cell activation. First, NK cells express multiple activating receptors, for example the NKG2D receptor that recognizes host stress-induced activating ligands on the surface of the virally infected or tumor cell. Second, NK cells are activated when the inhibitory receptors are not engaged. One such inhibitory receptor is NKG2A, which recognizes the non-classical

22 1. Introduction

MHC class I molecule HLA-E. Importantly, the inhibiting human KIR receptors recognize classical MHC class I molecules present on healthy cells (HLA-A, -B, and -C), and they stop NK cell inhibition in the absence of those ligands. This concept is called the missing self-recognition (38). NK cells are also activated by non-self ligands on the host cell surface, for example via the NKp46 receptor that recognizes viral proteins in both human and mouse. Additionally, NK cells express the low-affinity immunoglobulin G receptor FcγRIII (CD16) that allows targeting of cells coated with antibodies, which leads to antibody-dependent cellular cytotoxicity (ADCC); and toll-like receptors (TLRs) that detect pathogen-associated molecular patterns (PAMPs) and damage-associated molecular pattern (DAMPs). Lastly, NK cells express receptors that bind regulatory cytokines such as interferons and interleukins (37,39–44). To highlight the diversity of the stimuli recognized by NK cells, some of the human NK cell receptors and their ligands are listed in table 1-2 (45–48).

Table 1-2. Human NK cell activating and inhibiting receptors (45–48)

Receptor Function Receptor Ligand Additional information on family name ligand function

NCR Activating NKp46 HSPG, heparin Heparan Sulfate (Natural Proteoglycans, cell surface Cytotoxicity co-receptors, deregulated in Receptor) some tumors NKp44 NKp44 ligands Molecules exposed at the cell surface in tumors of during viral , i.e. influenza virus hemagglutinin NKp30a, b B7-H6 B7-H6 is a protein selectively expressed in several types of tumors Inhibitory NKp44 PCNA Proliferating cell nuclear antigen NKp30c ? ? NKG2 Activating NKG2D NKG2D ligands Stress induced ligands [1.4] (Natural Killer CD94/NKG2C HLA-E Non-classical MHC class I, Group 2) presents signal peptides from Inhibitory CD94/NKG2A HLA-E classical MHC class I CD94/NKG2E molecules CD94/NKG2F KIR Activating 2DS1/S2/S4 HLA-C/? Human leukocyte antigen, (Killer-cell display, display peptide 3DS1 ? immunoglobulin- fragments of intracellular like receptor) Inhibitory 2DL1/L2/L3 HLA-C proteins to cytotoxic T cells 3DL1/L2/L3 HLA -A, -B

23 1. Introduction

Receptor Function Receptor Ligand Additional information on family name ligand function

Adhesion Activating DNAM-1 PVR, nectin-2 PVR, poliovirus receptor, also molecules (CD226) called CD155; and Nectin-2; both overexpressed in some Inhibitory TIGIT PVR, nectin-2 types of tumors TACTILE FcgRIII Activating CD16 Fc IgG CD16 binds to the Fc portion of IgG antibodies

Activation of a single NK cells depends not only on the balance between multiple activating and inhibiting signals, but also on the maturation stage of the cell, and on the cytokine environment regulated by the neighboring immune cells. The complexity of NK cell regulation is based on the fine tuning of multiple receptors; it allows efficient killing of tumor cells or virally infected cells, and at the same time limits the hyper- responsiveness and possible self-reactivity of NK cells (37, 49).

Importantly, the exposure to different pathological conditions regulates NK cell maturation and NK cell repertoire, resulting in the generation of NK cell subsets with memory properties. Antigen-specific memory NK cells can be generated during exposure to some viruses and haptens. Additionally, certain inflammatory cytokine environments might also create non-antigen-specific memory NK cells with distinctive long-lasting effector functions. Both types, antigen-specific and non-antigen-specific memory NK cells, exhibit their properties after adoptive cell transfer (50).

1.2.3.2 Regulation of NK cell activity The NK cell response is controlled by the cytokine microenvironment and interactions with other immune cells, like T cells, macrophages and dendritic cells. Main regulators of NK cell proliferation and effector functions are type I interferon and interleukins (IL), i.e., IL-2, IL-12, IL-18, and IL-15. NK cell subsets with low killing capacity require priming for full activation (similarly to T cells). One of the main priming agents is IL-15, which is obligatory for the NK cell survival (51, 52).

1.2.3.3 Cytotoxic granule-mediated killing There are two main mechanisms of NK cell-mediated killing, the of cytotoxic granules and death receptor-mediated apoptosis (53).

24 1. Introduction

Cytotoxic granules released by NK cells have properties similar to and contain granzymes, perforin, and granulysin (the latter only in humans). Granzymes are related to serine proteases. They have many substrate and thus can activate cellular caspases and pro-apoptotic proteins, disrupt ER and mitochondrial membranes, and fragment DNA. Perforins disrupt the plasma membrane of target cells by creating pores with a diameter of 13 to 20 nm, or larger, depending on the perforin oligomerization. Granulysin is a cytolytic protein with a pore-forming activity that plays a role in protection against tumours and microbes like bacteria, fungi, and parasites (51, 53–55).

Multiple mechanisms prevent the lysis of NK cells themselves by their own lytic molecules. Granzymes are expressed as a zymogens; in the granules, they are stored in their active form, and they are inhibited by the low pH. The activity of perforins is regulated by their oligomerization state, pH, and Ca2+. It has been suggested that during transport to the granules, they are expressed as zymogens, or that their activity is regulated by association with an ER chaperone, calreticulin. The synthesis and trafficking of granulysins is not well studied (51, 53-55).

Killing of target cells is a multistep process initialized by integrin-mediated adhesion to the target cell and formation of the immunological synapse. The lytic granules are transported along the microtubules and fused with the NK cell plasma membrane due to the activity of various proteins including small GTPases and the SNARE complex. The neutral pH of the extracellular space activates the perforins and granzymes. Upon oligomerization, perforins form the pores that allow transport of granzymes into the target cells. Alternatively, granzymes can be endocytosed by a target cell (51, 53-55).

There are few proposed mechanisms of NK cell self-protection during release of the cytotoxic granules, including expression of granzyme inhibitors; inhibition of perforins by CD107a/LAMP-1 (Lysosomal-Associated Membrane Protein-1), which is also a degranulation marker; specific lipid composition of the membrane that reduces perforin intercalation; and expression of cathepsin B that inactivates perforin (51, 53-55).

1.2.3.4 Death receptor-mediated killing Engaging of the target cell death receptors can be mediated via the three types of NK cell receptors; first, TNF receptor (TNFR, also called CD120) that recognizes

25 1. Introduction tumour necrosis factor alpha (TFNa); second, Fas receptor (also called apoptosis antigen 1, or CD95) activated by Fas ligand (FasL); and third, TRAIL receptor (also called death receptor), specific for TNF-related apoptosis-inducing ligand (TRAIL) (51, 53).

NK cells stimulated by the activating signals or interleukins upregulate TNFa, FasL, and TRAIL. All those ligands are stored in the intracellular granules or vesicles within the NK cell cytoplasm, but it is unclear whether one vesicle contains only one type or multiple types of molecules. Once present on the NK cell surface, TNFa, FasL and TRAIL can bind to respective receptors of the target cell and activate death receptor- mediated apoptosis (51, 53).

1.2.4 NK cell killing kinetics Killing mediated by release of the cytotoxic granules is much faster than by activation of the death receptors (minutes vs. hours). It has been suggested that the cytotoxic granules can be mobilized and released faster than granules containing death receptors ligands. Alternatively, different intensity of the NK cell stimulation results in activation of different killing pathways. It is unclear which of the pathways is more efficient (51, 56).

One NK cell can kill multiple target cells; however, the immunological synapse is formed with one target at the time. In other words, NK cells kill their target cells one by one. As little as two to four cytotoxic granules are sufficient to initiate the killing, and during one killing event, an NK cell releases about 10% of its cytotoxic granules (51, 53, 56).

1.3 NKG2D receptor The NKG2D receptor (NKG2DR) is one of the central NK cell activating receptors, and the NKG2D ligand-receptor axis is alike between human and mouse. In the following chapters, both, the receptor and the ligands will be described (57).

1.3.1 NKG2D receptor structure The NK group 2 member D (NKG2D) receptor, also called NKG2D receptor, belongs to the C-type lectin CD94/NKG2 family of receptors. The protein is encoded by Killer Cell Lectin Like Receptor K1 (Klrk1) gene, which is a part of the NK gene complex (NKC) expressed on 12 in humans or in mice (57,58).

26 1. Introduction

The receptor is a type II transmembrane protein, expressed as homodimer connected by a disulfide bond. The intracellular domains do not have signaling properties, and each of the monomers is associated with a signaling adaptor molecule called disulphide adaptor molecule (DAP), DAP10 or DAP12 (both monomers associate with the same type of adaptor molecule at the time). DAP12 contains an immunoreceptor tyrosine-based activation motif (ITAM), whereas DAP10 contains the tyrosine-based motif (YINM) with co-stimulatory properties (57, 59).

1.3.2 NKG2D receptor expression NKG2DR is expressed on cytotoxic immune cells: mainly, and on almost all of the NK cells, but also on some subsets of CD8+ T cells, NKT cells, gdT cells, and activated macrophages. T helper/CD4+T cells generally lack NKG2DR, unless in some pathological conditions such as cytomegalovirus infection, juvenile-onset lupus, or Crohn’s disease (60, 61).

NKG2DR expression is upregulated in NK cells upon IL-15 treatment, in CD8+ T cells after activation of the T cell receptor (TCR), and in macrophages upon activation with lipopolisacharide (LPS). Stimulation of the NKG2DR in NK cells results in the production of IFN-g, TNFa, granulocyte-macrophage colony-stimulating factor (GM-CSF), and pro-inflammatory chemokines; and in activated macrophages, in the production of nitric oxide and TNFα. While in NK cells and macrophages, NKG2DR alone can lead to their activation (primary recognition), in T cells it has a co-stimulatory function during the TCR-dependent response (57, 59–61).

Importantly, in NK cells, NKG2DR is expressed starting from the early developmental stages, and its expression has a regulatory role in NK cell education. NKG2DR influences expression and responsiveness of other NK cell receptors and NK cell proliferation. NK cells isolated form Klrk1-/- mice divide faster, develop into altered subpopulations, are more likely to undergo apoptosis, are less responsive to tumors, and are hyper-responsive towards virally infected cells; most likely, all these phenotypes are due to general NK cell dysregulation (61, 62).

1.3.3 NKG2D receptor isoforms and function As a result of of the Klrk1 transcript, two isoforms of the receptor have been identified: a short isoform expressed in mouse (NKG2D-S), and a long

27 1. Introduction isoform expressed in both human and mouse (NKG2D-L). The cytoplasmic domain of NKG2D-L is 13 amino acids longer, whereas the transmembrane domain and ectodomain are the same between the isoforms. NKG2D-S can form a complex with the adaptor molecules DAP10 or DAP12, whereas NKG2D-L associates only with DAP10. Both NKG2D isoforms are expressed in NK cells, macrophages, and CD8+ T cells; however, the adaptor molecule DAP12 is only expressed in NK cells and macrophages, and it is absent in CD8+ T cells. Expression of NKG2D isoforms in NK cells also depends on the cell maturation stage. Freshy isolated, naïve NK cells express mostly NKG2D-L, whereas activated NK cells express NKG2D-S (59, 61).

NKG2DR engagement results in phosphorylation of either DAP12 or DAP12 tyrosine- based motifs and in activation of signaling pathways that lead to cytokine production or cytotoxicity. DAP12 phosphorylation stimulates signaling via SYK and ZAP70 tyrosine kinases, whereas DAP10 phosphorylation stimulates the phosphatidylinositol 3-kinase (PI3K) pathway (Table 1-3). Upon ligand binding, NKG2DR is internalized, most likely to avoid NK cell hypersensitivity (57, 61).

Table 1-3. NKG2D receptor isoforms (57).

NKG2D Species Adaptor Adaptor Activated signaling Properties isoform molecule molecule pathway signaling motif

NKG2D-L Human DAP10 YXXM PI3K Co-stimulation Mouse NKG2D-S Mouse DAP10 YXXM PI3K Co-stimulation DAP12 ITAM SYK or ζ-chain- Primary associated protein 70 activation kDa (ZAP70)

1.4 NKG2D ligands

1.4.1 NKG2D ligand families NKG2D ligands (NKG2DL) are stress-induced proteins, also called stress ligands, that are not expressed, or expressed at very low levels, in healthy cells. In humans, they consist of two families, MIC and ULBP/RAET. These include two MHC class I polypeptide–related sequences A and B (MICA, MICB) and six UL16-binding proteins, ULBP1, 2, 3, 4, 5 and 6, also known as retinoic acid early transcripts (RAET). Mouse

28 1. Introduction

NKG2DL consists of three subfamilies: MULT-1; H60a, b, and c; and RAE-1 with five isoforms, a, b, g, d, and e (Table 1-4) (63, 64).

1.4.2 NKG2D ligand structure All NKG2DL are structurally homologous to MHC class I molecules, with a similar Ig-like domain organization (Figure 1-6., [1.7.3]). Their extracellular part consists of a1a2-like domains and – only in MICA and MICB – an a3-like domain. Unlike MHC class I, NKG2DL do not bind antigenic peptide or b2m. Their membrane attachment is either a transmembrane domain or glycosylphosphatidylinositol (GPI) anchor. While in all complexes the arrangement of NKG2DL and NKG2DR domains is similar, there are significant differences in their binding affinities (Table 1-4) (65–69).

Table 1-4. Human and mouse NKG2D ligands (65-69).

Ligand Full name Gene Membrane Affinity to the name anchorage NKG2D receptor [M]

Human

MICA MHC class I polypeptide-related sequence A PERB11.1 TM 0.9-1 x 10-6 MICB MHC class I polypeptide-related sequence B PERB11.1 TM 0.8 x 10-7 ULBP1 UL16-binding protein 1 RAET1I GPI 1.1 x 10-6 ULBP2 UL16-binding protein 2 RAET1H GPI - ULBP3 UL16-binding protein 3 RAET1N GPI - ULBP4 UL16-binding protein 4 RAET1E TM - ULBP5 UL16-binding protein 5 RAET1G TM - ULBP6 UL16-binding protein 6 RAET1L GPI -

Mouse

MULT-1 Murine UL16-binding protein-like transcript-1 Ulbp1 TM 6 x 10-6 H60a Histocompatibility antigen 60a H60a TM 3 x 10-8 H60b Histocompatibility antigen 60b H60b TM 3 x 10-7 H60c Histocompatibility antigen 60c H60c GPI 9 x 10-6 RAE-1a Retinoic acid early-inducible protein 1-alpha Raet1a GPI 7 x 10-7 RAE-1b Retinoic acid early-inducible protein 1-beta Raet1b GPI 2-3.5 x 10-7 RAE-1g Retinoic acid early-inducible protein 1-gamma Raet1c GPI 5-6 x 10-7 RAE-1d Retinoic acid early-inducible protein 1-delta Raet1d GPI 0.5-7 x 10-7 RAE-1e Retinoic acid early-inducible protein 1-epsilon Raet1e GPI 3 x 10-8

29 1. Introduction

Although all NKG2DL share similar structural topology, there are significant differences in the amino acid sequence similarity. ULBPs share about 50% amino acid sequence similarity between each other, and about 22-25% with MICA and MICB. The similarity between human and mouse NKG2DL, and between different mouse subfamilies is only around 15-20%. On the other hand, different RAE-1 isoforms share between 88,5 and 92% of amino acid sequence identity. For comparison, mouse and human NKG2DR shares 69% of the amino acid sequence (63, 70, 71).

1.4.3 NKG2D genes NKG2DL genes are encoded on chromosome 6 in human, with MIC genes being located within the MHC gene region, and on chromosome 10 in mouse. While classical MHC class I molecules are present in all classes of jawed vertebrates, NKG2DL are present only in in the placental and marsupial mammals. NKG2DL genes are one of the most polymorphic, with at least 100 alleles of MICA and 40 alleles of MICB (the data are limited by the small sample number) (63, 72).

Single nucleotide polymorphisms (SNPs) are the main source of genetic variants in the a1a2-like domains and the a3-like domain-encoding regions. Additionally, short tandem repeat (STR) polymorphism occurs mainly in the transmembrane-encoding region. Interestingly, NKG2D ligands that are normally expressed with a transmembrane domain can become GPI-anchored proteins as a result of an STR . The lowest genetic variation occurs in the regions that encode those parts of the protein that interact with the NKG2DR (72, 73).

1.4.4 NKG2D ligand-receptor binding Both ectodomains of the NKG2DR homodimer are involved in ligand recognition.

Each of the monomers binds to one side of the ligand, a1- or a2-like domain, also called half-sites of the ligand (Figure 1-6., [1.7.3]). The receptor binding sites of different ligands overlap, and this region consists partially of b5’-b5 loop, also called the stirrup loop. In order to sustain a high affinity for diverse ligands, all of which are binding to the same receptor region, the NKG2DL interfaces have similar shapes, and the interface of the receptor is flexible. This allows the NKG2DR homodimer to position its binding site over the ligand half-sites. The receptor stirrup loop adapts its conformation upon the binding of the different ligands, and it can move towards or

30 1. Introduction away from the center of the complex. Additionally, there are certain ligand-contacting side chains or “binding hotspots” that are asymmetrically arranged across NKG2DR homodimers (65).

1.4.5 NKG2D ligand expression To ensure absence of the NKG2DL on the surface of healthy cells, their expression is regulated by multiple mechanisms during all steps of protein production. Different forms of cellular stress, for example infection, malignant transformation, oxidative stress, or ionizing radiation, induce transcription of the NKG2DL genes. Cellular processes that control expression of these genes are chromatin remodeling, heat shock response, ER stress, and the unfolded protein response. The regulation may be specific for a particular ligand, or allelic variant. NKG2DL are expressed with different tissue specificity, and during cellular stress, they are upregulated with various kinetics (72, 74, 75).

Regulation of NKG2DL expression plays a key role in the elimination of tumor and virally infected cells. Therefore, the NKG2D ligand-receptor axis is often hijacked in these settings. Consequently, most of the research literature describes NKG2DL regulation in the context of tumor or infection, and often it remains unclear how the regulation works in healthy cells. Most probably, the same regulation mechanisms that are disturbed in sick cells are used for a fine tuning of NKG2DL expression in healthy cells or during mild stress. Each of the steps that antagonize NKG2DL induction might work up to a certain threshold of the inducing factor. Since interference with NKG2DL regulation is a common immune evasion strategy of tumors, these processes are often targets of cancer (64).

1.4.5.1 Regulation of gene expression Molecules involved in the transcription of NKG2DL genes are, among others, NF-kB (Nuclear Factor kappa-light-chain-enhancer of activated B cells), the major regulator of the innate and adaptive immune response, and the transcription factor p53 that selectively regulates ULBP1 and ULBP2 (76). The DNA damage sensor pathway STING-IRF3-TBK1 induces RAE-1 in response to cytosolic DNA in tumor cells [1.6] (77). It is unclear how the splicing variants of the NKG2DL genes are regulated, but different splicing isoforms were described for MICA, ULBP4, and ULBP5 (72).

31 1. Introduction

1.4.5.2 Posttranscriptional regulation Posttranscriptional regulation of NKG2DL is strictly controlled by the mRNA destabilizing factor AUF1 (AU-rich element RNA-binding protein 1). During cellular stress, EGFR (epidermal growth factor receptor) pathway induces relocation of AUF1 from the nucleus, allowing translation of the NKG2DL mRNA (78).

NKG2DL transcripts are also regulated by multiple RNA binding proteins (RBPs), that tune mRNA stability, trafficking, and translation (79). For example, ULBP2 and MICB mRNAs are targets of the RNA-binding protein IMP3 (insulin-like growth factor 2 mRNA-binding protein 3), an oncogene upregulated in multiple types of cancer (80).

Endogenous/host microRNAs also control NKG2DL mRNA, and they are often upregulated in tumors leading to NKG2DL downregulation, and thus tumor progression. These types of microRNAs are also called metastasis-associated miRNAs (metastamiRs), and one example is miR-10b, which specifically down-regulates MICB and leads to reduced NK cell killing (72, 81, 82).

1.4.5.3 Posttranslational regulation The posttranslational regulation of NKG2DL includes secretion of soluble molecules by alternative splicing, exosome secretion, and cleavage (shedding) by cellular proteases. Release of soluble NKG2DL is an immune evasion strategy in many types of tumors, and high serum concentrations of shed NKG2DL, for example MICA, are correlated with tumor progression and poor therapy prognosis(83). Lack of NKG2DL on the surface of tumors limits immune recognition, while chronic engagement of the NKG2DR by soluble NKG2DL results in receptor internalization and reduced NK cell responsiveness (61, 72).

A disintegrin and metalloproteases (ADAM) proteases and matrix metalloproteinases (MMPs) are responsible for NKG2DL shedding at the cell surface, and some allelic NKG2DL variants with an altered transmembrane domain remain resistant to this process (83,84). NKG2D ligands are also components of exosomes secreted by tumors. Such exosomes also contain molecules that control angiogenesis, cell migration and immune response, and exosome secretion is a common growth- promoting strategy (85). Some ligands are more sensitive to these processes than others. For example, comparison between ULBP2 and ULBP3 suggests that the first

32 1. Introduction is more susceptible to metalloprotease-mediated shedding, while the latter is released in exosomes (86).

Apart from cell surface shedding, the trafficking and degradation rate of NKG2DL are also regulated by processes that remain to be characterized. For example, MICA was reported to be retained in the early secretory pathway and rerouted for the proteasomal degradation in melanoma cells by an unknown mechanism, while ULBP1 has a shorter half-life at the cell surface, and after internalization, it is more susceptible to degradation than other GPI-anchored ULBPs (87, 88).

1.4.6 NKG2D ligands diversity and expression patterns It remains unclear why cells express such a wide array of NKG2DL. One explanation suggests that pathogens, especially viruses with their multifaceted immune evasion strategies, put an evolutional pressure on NKG2DL diversity (or the other way around). NKG2DL variety may be also linked to their expression patterns during different kinds of cellular stress; affinity to the NKG2DR, and thus, ability to trigger NK cell response; and tissue specificity, with MICA being the least, and ULBP6 the most tissue-specific (Table 1-5). The above-mentioned explanations do not contradict each other. However, it is evident that the NKG2D ligand-receptor axis plays a role in both adaptive and innate immune responses, and therefore must be precisely regulated (61, 73, 79).

Table 1-5. NKG2D ligand RNA expression in different human tissues (89).

MICA MICB ULBP5 ULBP6 ULBP2 ULBP1 Organ ULBP3 ULBP4

amygdala 3,0 0,8 2,0 4,5 1,0 0,2 0,3 1,1 basal ganglia 4,3 0,8 0,6 2,3 1,2 0,2 0,3 0,1 cerebellum 3,0 3,0 13,9 5,3 0,3 0,2 0,2 0,0 cerebral cortex 6,8 1,0 1,5 6,5 2,0 0,2 1,0 0,2 corpus callosum 4,4 0,9 0,2 0,0 0,4 0,2 0,5 0,0 hippocampal formation 3,9 1,0 1,5 4,4 1,3 0,2 0,4 0,0 hypothalamus 1,0 0,6 0,3 1,0 2,3 0,0 0,2 0,0 midbrain 4,3 1,2 0,3 1,0 0,7 0,2 0,3 0,0 olfactory region 3,2 0,8 0,0 2,1 0,6 0,2 0,1 0,0 pons and medulla 12,6 1,2 1,0 3,9 0,8 0,2 0,5 0,0 spinal cord 7,9 1,5 1,2 0,6 0,5 0,2 0,9 0,0 substantia nigra 4,3 1,2 0,3 1,0 0,7 0,2 0,3 0,0

33 1. Introduction

MICA MICB ULBP2 ULBP3 ULBP4 ULBP5 ULBP6 Organ ULBP1 thalamus 3,4 1,1 0,2 0,1 0,3 0,3 0,2 0,0 retina 13,8 1,1 0,5 0,3 1,7 0,2 0,8 0,0 adrenal gland 9,7 1,2 0,6 1,7 3,6 0,5 10,0 0,1 parathyroid gland 13,4 0,2 0,6 0,3 1,9 0,1 0,1 0,0 pituitary gland 2,8 0,6 0,6 1,1 0,7 0,2 1,2 0,0 thyroid gland 8,7 2,4 3,8 3,5 8,2 0,2 0,7 0,0 esophagus 6,9 2,1 0,8 10,8 3,6 39,7 21,2 31,4 salivary gland 6,5 1,9 1,0 1,5 1,2 7,0 3,9 4,5 tongue 10,2 0,7 0,6 4,6 5,3 19,8 18,4 25,1 colon 9,5 1,6 0,7 0,6 2,8 0,3 0,6 0,1 duodenum 0,7 1,0 0,4 0,9 1,4 0,2 0,2 0,0 rectum 5,6 1,9 0,4 0,7 1,1 0,3 0,1 0,1 small intestine 7,6 5,0 0,3 0,4 1,0 0,3 0,5 0,0 stomach 6,2 1,4 0,9 0,7 2,9 0,2 0,6 0,0 gallbladder 13,1 1,7 0,5 1,6 4,4 0,2 1,2 0,1 liver 7,5 1,3 0,9 1,7 2,6 0,2 0,2 0,0 kidney 19,1 2,9 1,2 1,9 5,5 2,8 2,3 0,1 urinary bladder 6,3 3,2 0,7 4,1 1,8 1,3 2,8 0,2 ductus deferens 16,9 0,0 0,0 0,0 1,1 0,2 1,4 0,0 epididymis 14,7 1,3 0,5 0,5 2,1 0,6 4,3 0,0 prostate 12,6 1,1 0,8 0,6 1,3 0,4 2,9 0,1 seminal vesicle 2,1 0,7 0,2 0,4 0,4 0,2 2,2 0,1 testis 24,6 2,3 10,5 3,6 24,3 0,5 0,5 0,1 breast 8,3 3,5 0,5 5,9 8,2 1,4 2,5 0,3 cervix, uterine 11,0 1,8 0,4 1,6 1,9 10,4 5,6 6,1 endometrium 9,0 2,1 0,5 0,6 1,1 0,8 0,8 0,3 fallopian tube 7,9 1,1 0,9 0,5 3,6 0,6 0,5 0,0 ovary 15,1 1,3 1,2 0,2 2,0 0,6 1,9 0,0 placenta 4,3 0,8 1,3 4,4 1,4 0,5 0,1 0,0 vagina 11,9 26,5 4,9 5,2 3,1 12,6 5,8 8,8 heart muscle 10,8 2,2 1,2 4,1 1,2 0,2 0,9 0,0 skeletal muscle 6,2 0,8 1,6 0,6 0,7 0,7 0,4 0,1 smooth muscle 3,7 1,7 0,3 2,0 0,8 0,3 1,8 0,1 appendix 7,0 9,1 0,4 1,9 0,4 0,5 0,6 0,0 bone marrow 0,7 6,1 0,5 4,3 0,2 0,0 0,0 0,0 lymph node 6,5 7,8 0,2 1,1 0,3 0,2 0,1 0,0 spleen 7,7 11,7 0,3 0,5 0,5 0,6 0,2 0,0 thymus 1,2 8,1 0,2 0,5 0,3 0,3 0,2 0,7 tonsil 3,4 8,1 0,5 2,4 2,1 22,5 10,7 15,3 B-cells 1,7 2,2 0,7 0,0 0,3 0,6 0,0 0,0

34 1. Introduction

MICA MICB ULBP1 ULBP2 Organ ULBP3 ULBP4 ULBP5 ULBP6 dendritic cells 1,8 2,1 0,1 0,0 0,0 0,4 0,0 0,0 granulocytes 4,5 4,1 0,9 0,0 0,0 2,3 0,0 0,0 monocytes 1,9 4,8 1,8 0,0 0,0 0,6 0,0 0,0 NK-cells 0,3 1,9 0,3 0,8 0,0 0,5 0,0 0,0 T-cells 2,5 4,4 3,5 5,5 2,7 0,7 0,6 0,6 total PBMC 1,7 1,9 0,3 0,9 0,5 0,1 0,0 0,1 lung 15,3 6,9 9,7 6,2 3,1 0,6 1,3 0,2 pancreas 10,9 2,9 0,4 1,6 1,1 0,2 0,1 0,1 skin 5,4 1,0 0,6 1,0 1,0 14,6 4,3 2,6 adipose tissue 10,4 3,3 0,7 3,3 8,0 1,6 2,5 0,0

1.5 RAE-1 RAE-1 an NKG2D activating ligand, and its modulation by the MCMV is the subject of this thesis; therefore, it will be characterized further in the following chapter.

1.5.1 Expression, structure and function Originally, RAE-1 was identified as one of the genes upregulated in mouse embryonal carcinoma F9 cells upon retinoic acid treatment that would induce differentiation of these cells into endoderm-like cells. Because it was present in the differentiating embryonic cells, and not in the adult tissues, RAE-1 was thought to be involved in the early embryo development (90, 91). Further studies identified RAE-1 upregulation in virally infected cells and tumors, but not during heat shock treatment. Importantly, RAE-1 signaling alone in vivo can induce NK cell-mediated killing of tumor cells that express MHC class I molecules that act as an inhibitory NK cell signal (92, 93).

RAE-1, like other NKG2DL, is an MHC class I-like molecule with a structural homology to the a1 and a2 domains of MHC class I, but lacking an a3 domain equivalent (Figure 1-6., [1.7.3]). The polypeptide chain is 253 amino acids long, and it has five putative N-glycosylation sites and three putative O-glycosylation sites. The N terminus contains a 28 amino acid long signal peptide that is removed during the translocation into the ER. RAE-1 is a GPI-anchored protein, and during maturation, the C-terminal 24-amino acid long pro-peptide is cleaved in the process of serine lipidation (Figure 1-2) (90–92).

35 1. Introduction

Figure 1-2. RAE-1g amino acid sequence. Grey font, signal sequence; yellow font, lipidation site (attachment of the GPI anchor); blue font, pro-peptide (Uniprot entry O08604).

There are five isoforms of RAE-1 called RAE-1a, b, g, d and e, and these isoforms are specifically expressed in different mouse strains; for example, BALB/c mice express RAE-1a, b, and g, whereas C57BL/6 mice express RAE-1d, and e (93).

1.5.2 GPI anchor GPI anchor is the only glycolipid type of posttranslational protein modification. The common backbone of all GPI anchors consists out of ethanolamine phosphate, three mannoses, one non-N-acetylated glucosamine, and inositol phospholipid. This form of protein attachment is not exclusive to any protein family as it occurs in proteins with diverse functions including enzymes, receptors, protease inhibitors, and complement system proteins. Before GPI anchor attachment, the pre-proproteins are translocated into the ER lumen via the translocon complex, and they are believed to be attached to the membrane via their C-terminus. After cleavage of the propeptide, the GPI anchor is attached to the region with the characteristic GPI anchor attachment signal sequence, and the process is controlled by the GPI transamidase complex. The GPI anchor undergoes multiple modifications during the protein transport via the secretory pathway, therefore, the precise composition and molecular weight of the glycolipid attached to specific protein are usually unknown. Typical cargo receptors for many GPI anchored protein are the p24 proteins [1.9]. GPI-anchored proteins are usually

36 1. Introduction associated with plasma membrane regions enriched in sphingolipids and cholesterol (detergent-resistant membranes also called lipid rafts). The GPI anchor does not cross the membrane (94–96).

1.5.3 Regulation of RAE-1 expression Expression of RAE-1 during MCMV infection requires activation of the PI3K pathway, one of the main pathways regulating cell proliferation, survival, and metabolism. One out of three catalytic subunits of the class I PI3K called p110a is essential for this process, and the same catalytic domain was shown to be involved in the regulation of MULT-1 expression, however, it did not have an effect on H60a expression. Besides regulation of RAE-1 gene expression, the PI3K pathway may be involved in the stabilization of the RAE-1 mRNA (97, 98).

Importantly, RAE-1 induction occurs specifically in the infected and not in the surrounding cells, it requires expression of the early viral genes, and it is independent of viral DNA replication. The DNA damage response is not required for the RAE-1 expression during MCMV infection (98).

In healthy cells, expression of RAE-1 is repressed by the HDAC3 (histone deacetylase 3), an enzyme belonging to the histone deacetylase superfamily that prevents gene expression. During MCMV infection, the viral protein m18 binds to the CK2 (casein kinase 2) serine/threonine-selective kinase involved in the cell cycle control and regulation of the circadian rhythm that activates HDAC3. As a result, m18 inhibits HDAC3 and thus enables expression of RAE-1. Importantly, HDAC inhibitors that upregulate NKG2D ligand expression are common among other herpesviruses, for example HCMV IE1 protein (99, 100).

Besides in infection and tumors, RAE-1 is also upregulated in proliferating cells (both gene and cell surface expression) such as fibroblast tissues, embryonic cells, and wounded skin. The effect was studied specifically for RAE-1e, and it depends on the E2F family of transcription factors that directly bind and activate the RAE-1e promoter. Proliferation specifically, and not DNA damage, apoptosis, or senescence induces RAE-1e expression. Importantly, the effect depends on the intensity of proliferation and cell type, and most probably, it harmonizes with other NKG2D regulating mechanisms. These studies suggest another role for the NKG2D ligands that is not related to the immune response. It remains especially intriguing that RAE-1 is

37 1. Introduction expressed in embryonic tissues that are free of NK cells, suggesting that it may be involved in early development. Moreover, the proliferation state induces expression of only some NKG2D ligands, like some isoforms of RAE-1, and human ligands like MICA/B and ULBP2, but not MULT-1 and H60 (101).

1.5.4 NKG2DR/RAE-1 structure The crystal structure of RAE-1b in complex with NKG2DR was elucidated already in 2002 (102). The RAE-1b ectodomain consists of two long, discontinuous, and roughly parallel a-helical domains that are placed above an eight-stranded antiparallel beta sheet platform (Figure 1-6., [1.7.3]). The two a-helices are closer to each other than their equivalents in the MHC class I structure, and they move towards each other. The movement of the protein core is restricted with an exception of the three flexible loops arranged around the platform domain. Two disulfide bonds are present in the RAE-1b structure, one that connects the two a helices, and second that connects the b1 and b2 strands in the a1 domain (102).

Comparison between human NKG2DR/MICA and mouse NKG2DR/ RAE-1b complexes indicates that the NKG2DR universally binds to the ligands diagonally, however, the orientation of the complex components is slightly different (Figure 1-6., [1.7.3]). Unlike MICA, upon receptor binding, RAE-1b does not undergo large conformational changes. Moreover, the interaction interface between MICA and NKG2DR is larger and more shape-complementary than in the NKG2DR/RAE-1b complex (shown with total buried solvent-accessible surface area and shape correlation statistics). The glycosylation of the RAE-1b and NKG2DR does not seem to play a role in the interaction between them (66, 102).

1.6 Cytomegalovirus immunoevasins Cytomegaloviruses share strategies of host immune response invasion, and many of them are focused on inhibiting T cell and NK cell activation. These strategies exploit host cellular pathways or mimic host signaling in order to limit immune activating signals and enhance immune inhibiting signals (7,103).

A large number of immunoevasins is dedicated to downregulation of host molecules expressed on the cell surface in order to activate immune responses. Among those, proteins that downregulate via MHC class I molecules are

38 1. Introduction especially important. Downregulation is usually accomplished by intracellular retention or degradation of the host protein. For example, HCMV US2 and US11 target MHC class I for proteosomal degradation by ER-associated degradation (ERAD) pathway, while MCMV m06 reroutes MHC class I molecules to the . HCMV US3 and MCMV gp40 retain MHC class I in the early secretory pathway [1.7.1]. Immunoevasins may also indirectly interfere with the maturation of MHC class I, such as US6 that blocks TAP transporter to prevent peptide loading (7, 103, 104).

Stabilization of inhibitors expressed on the cell surface is another strategy used by the virus. HCMV UL40 encodes a leader sequence that can be loaded into HLA-E in a TAP-independent manner. As a result, peptide-loaded HLA-E is stabilized and expressed at the cell surface where it inhibits NK cells via the inhibitory NKG2A receptor (105). MCMV m04 (gp34) binds MHC class I and escort them to the cell surface to avoid NK cell “missing self” recognition (106).

Cytomegalovirus also expresses inhibitors of immune response that mimic host proteins. HCMV UL18 is a MHC class I homologue that binds to the inhibitory NK cell receptor LIR1 (107). MCMV m157 bind to inhibitory and activating NK cell receptors, and thus provides protection from NK cell lysis of infected cells in some mouse strains (108). HCMV also encodes a homologue of anti-inflammatory interleukin 10 (IL10), vIL10, that suppresses expression of MHC class II (104, 109–112).

One important component that is inhibited by the virus is interferon signaling (113). The cGAS-STING signaling pathway upregulates inflammatory genes in response to cytosolic DNA. HCMV and MCMV infections trigger this pathway, resulting in increased expression of interferon. cGAS (Cyclic GMP-AMP synthase, cGAMP synthase) is a cytosolic DNA sensor. Upon DNA stimulation, cGAS synthesizes cGAMP (Cyclic guanosine monophosphate–adenosine monophosphate), also referred to as 2′,3′-cGAMP, which is an endogenous second messenger that induces STING (stimulator of interferon genes). Upon activation, ER-resident STING molecules dimerize and translocate to the Golgi, where they induce phosphorylation and downstream signaling via the TBK1 kinase (TANK Binding Kinase 1) and the transcription factor IRF3 (Interferon regulatory factor 3) (114, 115).

HCMV UL83 and UL31 proteins inhibit enzymatic activity of cGAS, and thus block the formation of the secondary messenger cGAMP. HCMV UL42 also prevents binding

39 1. Introduction of DNA to cGAS, and it interferes with trafficking of STING, which is also crucial for the interferon induction. In MCMV, an immunoevasin interfering with the cGAS-STING pathway is gp40 [1.7.3] (113, 116, 117)

Other strategies that improve viral fitness include inhibition of apoptosis, for example HCMV viral Inhibitor of Caspase-8 Activation (vICA) UL36; or disruption of the actin cytoskeleton of the target cell to obstruct formation of the immunological synapse with NK cells or T cells by HCMV UL135 (118–120). Finally, there are miRNAs that inhibit translation of stress ligands, for example HCMV miR-UL112, which inhibits translation of the NKG2DL, MICB (103, 104, 112, 121).

The next chapter will focus on the immunoevasins that target NKG2DL, and thus limit NK cell recognition. Many of these proteins have multiple roles in the inhibition of immune response, but the molecular mechanism of the described interactions is often poorly characterized (Figure 1-3) [1.6.1, 1.6.2].

Figure 1-3. Overview of human and mouse immunoevasins that target NKG2D ligands. Black font, NKG2D ligand; red font, immunoevasin; grey font, other non-NKG2D target. Arrows point towards the targets of specific immunoevasins [1.6.1; 1.6.2].

40 1. Introduction

1.6.1 Human cytomegalovirus immune evasion of NKG2D ligand-receptor pathway

1.6.1.1 UL16 One of the best-studied HCMV immunoevasins that target NKG2DR recognition is UL16, a glycoprotein that downregulates specifically cell surface MICB, ULBP1, and ULBP2 (70). UL16 binds to MICB (and presumably ULBP1 and ULBP2) via three contact regions, forming a “saddle-like” structure, and the binding sites between UL16 and MICB overlap with binding sites between NKG2DR and MICB (Figure 1-6.,

[1.7.3]). The a2 domain of MICB is crucial for the tight binding to UL16, and only seven amino acids differ between UL16-susceptible MICB and UL16-resistant MICA. The interaction between UL16 and MICB is independent of glycosylation of both proteins (122,123). UL16 also downregulates cell surface ULBP6, and both proteins were shown to colocalise in the cell interior during HCMV infection, although no further investigation of this interaction was conducted (74).

The cellular localization of UL16 remains unclear. In some reports UL16 was restricted to the ER, or the ER and cis-Golgi, where it stabilized and retained MICB (124–126). According to other studies, UL16 was present mostly in the ER and the trans-Golgi network (TGN), but also to some extent at the cell surface and inner nuclear membrane; it did not affect MICB maturation significantly; and both proteins colocalized in the ER and the TGN. Trafficking of UL16 was however very slow, and the cell surface internalization rate was fast, suggesting that only a small fraction of UL16 might be present at the cell surface at steady state (127). Such significant differences in protein maturation may arise from using different cell lines, UL16 expression systems, and the antibodies used for MICB detection (124–127).

The cytoplasmic tail of UL16 carries a YQRL motif that is involved in binding of adaptor proteins, and was proposed to be partially responsible for the retention properties of UL16. UL16-mediated downregulation of NKG2DL results in impaired NK cell-mediated lysis of infected cells at later stages of infection, and decreased T cell activation (124, 126).

1.6.1.2 UL142 UL142 is a heavily glycosylated protein that is expressed late during infection (74 hours post infection); it downregulates NKG2DL resistant to UL16, MICA and

41 1. Introduction

ULBP3; and is encoded by a highly polymorphic gene that is missing in the laboratory strains of HCMV (128–130).

UL142 is localized mostly in the ER and cis-Golgi, but some fractions are also detectable at the cell surface. The cell surface population of UL142 is however not detectable by Western blot or radioactive pulse chase, suggesting that it is only a small quantity; or, alternatively, glycosylation of UL142 remains EndoH sensitive after passing the Golgi compartment (131).

UL142 downregulates MICA and ULBP3 by retaining them in the cis-Golgi (129, 130). It lacks any known retention/retrieval signal, but the transmembrane and lumenal domains of UL142 are required for intracellular retention of MICA. The retention might be maintained either by an unknown retention signal in the UL142 sequence, or by an interaction with a cellular retention factor. Interestingly, the MICA*008 allele with an altered transmembrane domain and cytoplasmic tail is resistant to UL142, suggesting that these regions of both proteins are important for the interaction (130, 131).

1.6.1.3 UL148A UL148A downregulates full-length MICA at earlier time after infection (25 hours), compared to UL142. UL148A on its own is insufficient to control MICA. and it requires an additional, unknown viral factor. The interaction leads to lysosomal-dependent degradation of MICA, and results in reduced NKG2D-dependant NK cell killing (132).

1.6.1.4 US12 gene family The best-studied members of the US12 gene family are US18 and US20, immunoevasins that reduce the cell surface expression of MICA by rerouting it for lysosomal degradation. While co-expressed with US18 and US20, MICA becomes EndoH resistant, suggesting that the these immunoevasins control MICA in a post- cis-Golgi compartment. Interestingly, lysosomal blockage does not rescue MICA cell surface expression but results in its intracellular accumulation (75).

Deletion of a single immunoevasin, US18 or US20, from the HCMV genome does not have a significant impact on MICA cell surface level, but deletion of both results in MICA cell surface rescue, suggesting that they can partially compensate for each other’s function. Analysis of HCMV deletion mutants also suggest that US18 is subjected to a more rapid turnover than US20, and that it is involved in viral

42 1. Introduction replication. Downregulation of MICA by US18 and US20 results in protection of infected cells against NK cell lysis (75).

US18 and US20 also downregulate B7-H6, a ligand of an activating NK cell receptor NKp30. B7-H6 is upregulated in tumours and during inflammatory, for example HCMV infection, and therefore similarly to NKG2DL it is called a “stress ligand”. During infection, US18 and US20 target B7-H6 for lysosomal degradation, and thus decrease NK cell recognition of infected cells (133–135).

The US12 gene family consists of ten genes (US12-US21) involved in tropism, virion maturation, and immune evasion. Most US12 family members are not yet characterized. Proteomic analysis of cells infected with HCMV deletion mutants suggest that US12 family members control multiple host molecules crucial for the immune signalling and NK cell activation including cytokine receptors, cell adhesion molecules, and activating ligands like MICB and ULBP2 (134).

Even though each of the US12 family members is able to downregulate specific target to some extent, in most cases co-operation of multiple immunoevasins is the most effective. Moreover, each immunoevasin controls more than one host molecule, and UL18 and UL20 impact most known host molecules to some degree. Interestingly, US13 and US12 regulate UL16 (134).

1.6.1.5 US9 US9 is a transmembrane glycoprotein encoded in the US2-US11 genome region, and it targets the UL142-resistant MICA allele, MICA*008. The transmembrane domain and the cytoplasmic tail of MICA*008 are truncated due to a frameshift mutation, and as a result, it is attached by a GPI anchor instead of a transmembrane domain. The attachment of the GPI anchor to MICA*008 is slow compared to other GPI-anchored proteins; it takes about three hours, and it is mediated by a nonstandard and unknown pathway. For comparison, maturation of full-length MICA alleles takes about 30 minutes. MICA*008 is present mostly in detergent-resistant membranes, it is released in exosomes, and it is the most common allele in the human population (136).

US9 targets itself and MICA*008 for proteosomal degradation. Very careful maturation analysis revealed that US9 affects only the mature MICA*008 form, and that the unusual GPI anchoring of this allele is required for the interaction. A chimera

43 1. Introduction of MICA*008 with the GPI anchor attachment site of ULBP3 is resistant to US9, whereas a MICB chimera with the GPI anchor attachment site of MICA*008 is downregulated by US9. While the presence of US9 reduces the cell surface MICA*008 population, the overall intracellular level is unchanged. Additionally, during HCMV infection, the US9 effect on MICA*008 is relatively weak. These findings suggested that that there is an additional viral factor that controls immature MICA*008 and retains it in the ER (based on glycosylation analysis of the intracellular MICA*008 population). Downregulation of MICA*008 by US9 results in reduced NKG2D- mediated NK cell killing (136).

1.6.2 Mouse cytomegalovirus immune evasion of NKG2D ligand-receptor pathway The majority of the following immunoevasins belongs to the m145 gene family that contains ten members, and eight of those are predicted or proven MHC class I homologues (in bold): m144, m145, m146, m150, m151, m152, m153, m154, m155, m157 and m158 (137, 138).

1.6.2.1 m155 m155 is a glycoprotein that specifically downregulates H60 during MCMV infection by a proteasome-dependent mechanism, and thus impairs NKG2D-mediated NK cell recognition of infected cells. Both proteins form a complex, and trafficking analysis suggests that m155 impairs H60 beyond the early secretory pathway, most probably by posttranslational modification. During MCMV infection, H60 maturation and stability are unchanged, however H60 is undetectable at the cell surface, suggesting intracellular relocation or epitope alteration. Importantly, m155-mediated down-regulation of H60 is not complete in infected cells suggesting that there is another immunoevasin that downregulates this NKG2DL [1.6.2.3] (139, 140) m155 also downregulates the cell surface level of CD40, a co-stimulatory molecule expressed by antigen presenting cells to promote CD4+ and CD8+ T cell response. CD40 downregulation by m155 was documented in dendritic cells and macrophages (141).

Interestingly, during MCMV infection, m155 accumulates in the ER, whereas an ectopically expressed protein traffics beyond this compartment, suggesting that other viral factors regulate m155 localization (140, 141).

44 1. Introduction

1.6.2.2 m145 m145 downregulates cell surface MULT-1, and thus protects infected cells from NK cell recognition. MULT-1 glycosylation, maturation, and degradation rate are not influenced by m145 when both proteins are ectopically co-expressed and during MCMV infection. m145 relocates MULT-1 beyond the cis-Golgi compartment by an unknown mechanism (142).

1.6.2.3 fcr-1/m138 fcr-1 was first described as a protein with IgG binding properties, expressed as soon as two hours post infection, and throughout the whole MCMV replication cycle (143). Although fcr-1-deletion MCMV mutant growth was restricted in vivo, the IgG-binding property did not seem to be relevant for the viral replication, or for the antibody- mediated clearance of the virus (144). Later, fcr-1 was identified as a selective regulator of MULT-1, H60, and RAE-1e, that downregulates these host molecules independently of other viral proteins (145, 146).

The mechanism of cell surface downregulation seems to be different for each of these ligands. The predicted structure of fcr-1 consists of three Ig-like domains (Ig1, Ig2, Ig3). Ig1 alone is sufficient to downregulate MULT-1, whereas all Ig-like domains are required for the downregulation of H60. Fcr-1 reduces MULT-1 expression by interference with cell surface recycling and by enhanced lysosomal degradation. However, the same effect was not observed for the H60 (145). Additionally, fcr-1 downregulates cell surface forms of RAE-1e by modulation of the endocytosis rate. Interestingly, the closely related RAE-1d is not affected by this immunoevasin. Deletion of fcr-1 results in NKG2D-dependant attenuation of MCMV in vivo (146). fcr-1 also controls B7-1, that belongs to the B7 family of proteins expressed on antigen presenting cells and regulating T cell responses. Ectopically expressed in dendritic cells, fcr-1 downregulates cell surface B7-1, and thus impairs ability to promote T cell activation. fcr-1 impairs maturation of B7-1, and relocalizes it to the lysosome. Both proteins form a complex in the cells (147). Interestingly, when expressed alone, fcr-1 is in the early secretory pathway, whereas during MCMV infection, it reaches the cell surface (144, 147).

45 1. Introduction

1.7 gp40 gp40 is encoded by the m152 gene, and it is expressed during the early phase of infection. Transcription of m152 starts two hours post infection (hpi), and it reaches the maximum at four hpi. Protein expression starts three hpi, it reaches the maximum at five to six hpi. After 15 hpi, the expression decreases and is continuous at lower level throughout the replication cycle (24 hours). The polypeptide chain is 378 amino acid long, which corresponds to a molecular weight of about 43.5 kDa; it has three putative glycosylation sites, and it is embedded in the plasma membrane with a type I transmembrane domain (Figure 1-4). Two glycosylated forms, gp40 and gp37, are detected in fibroblast cells infected with MCMV, and inhibition of glycosylation results in the appearance of one 34 kDa signal (148).

10 20 30 40 50 MLGAITYLLL SVLINRGETA GSSYMDVRIF EDERVDICQD LTATFISYRE

60 70 80 90 100 GPEMFRHSIN LEQSSDIFRI EASGEVKHFP WMNVSELAQE SAFFVEQERF

110 120 130 140 150 VYEYIMNVFK AGRPVVFEYR CKFVPFECTV LQMMDGNTLT RYTVDKGVET 160 170 180 190 200 LGSPPYSPDV SEDDIARYGQ GSGISILRDN AALLQKRWTS FCRKIVAMDN

210 220 230 240 250 PRHNEYSLYS NRGNGYVSCT MRTQVPLAYN ISLANGVDIY KYMRMYSGGR 260 270 280 290 300 LKVEAWLDLR DLNGSTDFAF VISSPTGWYA TVKYSEYPQQ SPGMLLSSID

310 320 330 340 350 GQFESSAVVS WHRGHGLKHA PPVSAEYSIF FMDVWSLIAI GVVFVIVFMY

360 370

LVKLRVVWIN RVWPRMRYRL VYINCRVW

Figure 1-4. Amino acid sequence of gp40. Grey font, signal sequence; underlined font, linker; bold fort, transmembrane domain (Uniprot entry A8E1R8).

gp40 is a structural homologue of MHC class I, and it has Ig-like a1, a2 and a3 domains arranged in a comparable way (Figure 1-6., [1.7.3]). An a1a2 domain consists of the alpha 1 helix and the discontinuous alpha 2 helix oriented in an antiparallel way, and it is placed above a platform with of eight antiparallel beta strands. Similarly to NKG2D

46 1. Introduction ligands, gp40 does not bind peptide or b2m. There are two disulfide bonds, one that connects the N-terminal part of gp40 to the alpha 2 helix, and another within the a2 domain (149).

1.7.1 gp40 interaction with MHC class I gp40 was first identified as an MHC class I downregulating immunoevasin. It impairs antigen presentation to the cytotoxic T cells when expressed alone and during the MCMV infection in vitro and in vivo. In the presence of gp40, MHC class I molecules accumulate in the ERGIC and cis-Golgi (148, 150).

Further studies by Ziegler et al. have shown that the lumenal part of gp40 is sufficient to retain MHC class I molecules, although the efficiency is much higher with the full-length protein. gp40 overexpressed in fibroblast cells using vaccinia virus localizes in endosomal/lysosomal compartments, and does not co-localize with MHC class molecules I that remain retained in the pre-cis-Golgi compartment for up to 16 hours (151).

Similarly to other immunoevasins [1.6], the aforementioned studies failed to explain how exactly gp40 interferes with MHC class I maturation, and it was especially intriguing since both proteins appeared not to bind to each other, or to even colocalize in the same compartments of the secretory pathway. The interplay between these two proteins was investigated further by the research group of Prof. Sebastian Springer, and thanks to the deep knowledge of the MHC class I biology and excellent methodological approaches molecular details of gp40/MHC class I interaction are known, and can be used as a model to learn about other viral/host protein interactions (152, 153).

Janssen et al. show that gp40 has a slightly different impact on different MHC class I alleles. When co-expressed, H-2Db is more sensitive to gp40, and it is localised in the ER, ERGIC and cis-Golgi, compared to H-2Kb, which is present in the ERGIC and cis-Golgi. In their expression system (using retroviral transduction), most of gp40 itself is also retained in the early secretory pathway. Importantly, both proteins form a complex within the cell, and analysis using antibodies against different parts of the MHC class I molecules indicates that protein orientation in the gp40/MHC class I complex resembles the gp40/RAE-1g complex in that gp40 most probably binds to the top (membrane-distal) part of the a1a2 domain of MHC class I. The complex

47 1. Introduction circulates in the early secretory pathway, between ER, ERGIC and cis-Golgi, as shown by analysis of the acquisition of partial EndoH resistance in radioactive pulse-chase experiments. gp40 also co-precipitates with some components of the peptide loading complex (PLC), , calnexin and calreticulin; however, association with the PLC components does not play a role in gp40-mediated MHC class I retention, since it functions in all retention-deficient cell lines with deletions of individual PLC components; neither does gp40 impair peptide loading, as evidenced by the high thermostability (strong binding of high-affinity peptides) of the class I molecules retained in the early secretory pathway (152). gp40 does not have any known retention signal, and the part of gp40 that is essential for retention is the 43 amino acid long linker that connects the transmembrane domain with the lumenal domain. Replacement of the linker sequence with the random sequence (Gly4Ser)9 does not impair protein folding, but it results in a rapid progress of the gp40 linker mutant (gp40LM) to the trans-Golgi and subsequent degradation in the lysosomes. MHC class I molecules co-expressed with gp40LM are no longer retained, and they are restored at the cell surface. The gp40LM still binds to MHC class I, and both proteins still form a complex when they are trapped in the early secretory pathway after treatment with brefeldin A (which inhibits protein export from the ER) (152, 153).

Three quarters of the gp40 linker are necessary for the retention mechanism, which consists of gp40 binding to the p24 family of proteins. The strongest interaction is observed for the p24 member TMED10, followed by TMED9, TMED2, and TMED5. TMED10 carries retention and retrieval motifs, and therefore gp40 uses it as an ER anchor for retaining MHC class I. Interaction between gp40 and TMED10 is independent of binding to MHC class I, and MHC class I molecules themselves do not interact with any of the p24 proteins. Absence of TMED10 has the same effect as use of the gp40 linker mutant, i.e., the rescue of cell surface MHC class I (153).

1.7.2 gp40 interaction with RAE-1 Studies of the gp40/MHC class I interaction have indicated that gp40 modulates NK cell activation and the effect differs between the mouse strains (154). The target controlled by gp40 was identified as RAE-1 and depended on the RAE-1 isoform ( strains express different RAE-1 isoforms). gp40 downregulates cell

48 1. Introduction surface RAE-1 and thus interferes with the NKG2D-based NK cell activation in vitro and in vivo (93).

During MCMV infection, RAE-1d is mostly preserved on the surface, whereas RAE-1g is not. RAE-1g is downregulated already four hpi, and eight hpi, the majority of cells express no or very low levels of this isoform. RAE-1g accumulates almost entirely in the immature form that is retained in the ER. In contrast, RAE-1d is divided into two populations of mature and immature protein. The main difference between these two isoforms is the PLWY motif, which is present in RAE-1g and absent in RAE-1d; it might impact the glycosylation of RAE-1. RAE-1d with the PLWY insertion mimicks RAE-1g in that it is less stable in uninfected cells, and it does not mature during MCMV infection. gp40 does not form a complex with RAE-1 in the cells2 (155).

Qualitative and quantitative studies demonstrate that gp40 and RAE-1 form a direct noncovalent complex in vitro and the stoichiometry of components is 1:1. Those RAE-1 isoforms included in the study, i.e., RAE-1b and g, which contain the PLWY motif, and RAE-1d, which lacks it, bind to gp40; however, the interaction with RAE-1g is the strongest, while that with RAE-1d is the weakest (Table 1-6) (65–67, 149, 156).

Table 1-6. Affinity of RAE-1 isoforms to the NKG2D receptor and to gp40 (65-67, 149, 156).

RAE-1 Affinity to NKG2D Method and reference Affinity to Method and isoform receptor (M) gp40 (M) reference

RAE-1b 2 x10-7 SPR*, McFarland 2003 3 x 10-7 SV**, Zhi 2010 3.5 x 10-7 SPR, O'Callaghan 2001 0.9 x 10-7 SPR, Wang 2012 RAE-1g 5-6 x 10-7 SPR, O'Callaghan 2001 1 x 10-7 SV, Zhi 2010 0.43 x 10-7 SPR, Wang 2012 0.42 x 10-7 SPR, Wang 2012 RAE-1d 0.5 x 10-7 SPR, McFarland 2003 34 x 10-7 SV, Zhi 2010 7.0 x 10-7 SPR, O'Callaghan 2001 RAE-1e 0.03 x 10-7 SPR, 193 x 10-7 SPR, Wang Carayannopoulos 2002 *** 2012

* SPR, surface plasmon resonance; ** SV, Sedimentation velocity ultracentrifugation; *** RAE-1e in this publication is named RAE-1B6

2 This is contradicted by our own findings [4]

49 1. Introduction

The crystal structure of the gp40/RAE-1g complex resembles that of the NKG2D/RAE-1b complex ([1.5.4], and (Figure 1-6., [1.7.3]). gp40 binds to RAE-1g with two interfaces, a1a2 domain of gp40 on one side, and a3 domain on the other site of RAE-1g. The gp40/RAE-1g interaction surface is more shape-complementary than that of the NKG2D/RAE-1b complex (interface shape complementarity (Sc) value 0.68 vs 0.63) (149). It is unknown if there is a significant molecular adjustment of both proteins during complex formation. If one models the NKG2D/RAE-1g complex on the basis of the NKG2D/RAE-1b complex, then one can conclude that the interaction surfaces of the gp40/RAE-1g and NKG2D/RAE-1g complexes are very similar, and this suggests that gp40 and NKG2D might compete for binding to RAE-1g (Table 1-6) (Figure 1-6., [1.7.3]). (149).

Importantly, the crystal structure of the gp40/RAE-1g complex demonstrates that besides the PLWY motif in RAE-1, the interaction with gp40 also depends on the K154/E159 motif. Both motifs are either deleted or mutated in RAE-1e isoform (Figure 1-5) (149).

50 1. Introduction

Figure 1-5. Amino acid sequence alignment of RAE-1 isoforms. Red bracket, the PLWY motif; green lines, K154/E159 motif. Amino acid sequences were obtained from the Uniprot database, with entry numbers: RAE-1a O08602, RAE-1b O08603, RAE-1g O08604, RAE-1d D7F2B5, RAE-1e Q9CZQ6.

1.7.3 gp40 interaction with STING gp40 also antagonizes cGAS-STING signaling. When expressed in immortalized macrophages, gp40 targets cGAS-STING signaling and consequently reduces secretion of INF-b. gp40 does not impair activation or dimerization of STING, but it delays the trafficking of STING dimers to Golgi. Both proteins co-localize in the ER of unstimulated cells and in the Golgi of cells stimulated by cGAS expression. Presence of gp40 in the Golgi is only observed in cells co-expressing STING. In stimulated and unstimulated cells, both proteins form a complex via the N-terminal lumenal domain of gp40 and two lumenal loops of STING. Interestingly, gp40 linker region is not required for the interaction. gp40 does not recognize human STING, presumably because the lumenal loops of STING are not conserved.

51 1. Introduction

The significance of the gp40/STING interaction was also demonstrated during MCMV infection. In infected cells gp40 and STING form a complex, and STING translocation as well as phosphorylation of TBK1 and IRF3 are delayed (117).

Notably, during the early stages of MCMV infection, the NF-kB pathway is crucial for viral replication, and this pathway is also activated by STING. However, gp40-mediated inhibition of STING specifically impacts IRF3, and NF-kB signaling can still be triggered by ER-resident STING (before it translocates to the Golgi) (117).

52 1. Introduction

Figure 1-6. Crystal structures of NKG2D ligands, NKG2D receptor, HCMV and MCMV immunoevasins, and mouse MHC class I H-2Kb. From top left; RAE-1g top view with amino acids depicted in colors: PLWY motif (green), K154 (cyan), E159 (blue); RAE-1g bottom view with putative glycosylation sites (blue bonds); RAE-1b top view, ULBP6 top view, MICA top view, H-2Kb with ovalbumin-derived epitope SIINFEKL (SL8) (black bonds); b MICB top view; gp40 top view; MICA side view; H-2K with b2m (red); NKG2D dimer, each monomer depicted in different color (ochre and yellow 1), with the stirrup loops (blue bonds); gp40 side view; NKG2D (ochre)/MICA (pink) complex; NKG2D (ochre)/RAE-1b (magenta 1) complex; UL16 (yellow 2)/MICB (mauve) complex; gp40 (orange)/RAE-1g (magenta 2). Pictures prepared in VMD based on PDB entries: 4G59 (gp40/RAE-1g), 4PP8 (NKG2D/RAE-1b), 4S0U (NKG2D/ULBP6), 1HYR (NKG2D/MICA), 5OQF (H-2Kb), 2WY3 (UL16/MICB) (102,123,157–159).

53 1. Introduction

1.8 Models to study viral immune evasion There are different experimental systems to study immunoevasins and their functions3. The usual way of identifying the viral protein with a specific function, such as the downregulation of a host cell ligand, is an infection of cells with viruses that contain deletions of gene families and/or specific genes. A comparison of the effects of wild type and of deletion virus on the cellular levels of the host protein then allows to pinpoint a specific viral gene responsible for the effect. Usually, this is done by immunostaining and flow cytometry or by Western blotting, depending on the location of the target protein. However, these methods require screening for individual proteins, which is laborious, time-consuming, and requires good quality antibodies to detect the prospective targets. Recently, advances in mass spectrometry-based proteomic approaches have allowed a comprehensive simultaneous readout of all host proteins, both intracellular and at the cell surface, that are affected by infection with a specific virus. On the other hand, such methods require access to adequate mass spectrometry facilities and proteomic analysis experience.

Once the immunoevasin responsible for the specific immune-modulating effect is identified, detailed studies are performed to understand the molecular mechanism of this effect. It can be studied using cells that are infected with the wild type virus and others infected with control deletion viruses that do not contain the gene of interest. Additionally, the immunoevasin can be individually expressed in the cells. In both cases, the immunomodulated host protein might be an endogenous one, or if the expression levels are too low, it can be transfected into the cells.

Expression of a single immunoevasin allows to discover subtle effects and interactions with the host protein. Moreover, in such a system, it is easier to use mutated versions of the protein, and to identify those portions responsible for the observed effects, or binding between proteins. Transfections allow easier analysis of protein maturation and complex formation using SDS-PAGE-based methods, like radioactive labeling or immunostaining, but they often result in overexpression of proteins and do not represent the physiological conditions.

To learn about the protein structure and complexes, proteins can be expressed in bacterial, insect, or yeast cells. If in vitro expression and purification is successful,

3 The chapter is based on refs. (74, 75, 124-156) and discussion with Dr. Anne Halenius from the Institute of Virology in the University Medical Center Freiburg.

54 1. Introduction one can measure the binding affinity between complex components, for example with surface plasmon resonance, or use protein X-ray crystallography to resolve the structure.

Important though they are, all findings made with immunoevasins studied in cell culture or in vitro, should be further validated during actual infection. Viral proteins very often work collectively, and the effect of a single protein might be significantly altered when it is active during infection with other proteins. Additionally, upon infection, the expression of the host proteins is altered, for example down- or upregulated, and therefore, the ratio of the immunoevasin and its target can be correctly estimated only upon infection.

If possible, the relevance of the immune-modulating effect should be also confirmed in an actual infection scenario in vivo, because the infection rate differs between organs, and the immune response against the pathogen is complex and may depend on many types of immune cells. Alternatively, the response of isolated immune cells might be tested upon mixing with infected cells.

All these experimental settings complement each other and provide different levels of insight into immune evasion. Whenever choosing an experimental system, it is important to ask specific questions and to differentiate the aims set to understand the effect of the viral protein on the host organism or the molecular mechanism of interaction between the proteins.

1.9 The p24 proteins

1.9.1 Nomenclature p24 is a family of proteins originally isolated from COPI and COPII-coated (coat protein complex I and II) vesicles as components involved in vesicular transport (160,161). They are present in yeast, plants and mammals and divided into four subfamilies, α, β, δ, and γ, based on their . There are several nomenclature systems used in parallel to annotate p24 proteins, and their names used commonly in mammals are summarized in Table 1-7 (162).

55 1. Introduction

Table 1-7. Nomenclature of p24 proteins in mammals (162).

Subfamily Systematic name Systematic database name/ Alternative names gene name *

a p24a1 TMED11 ** gp25L p24a2 TMED9 p25, GMP25, p24a3 TMED4 GMP25iso b p24b1 TMED2 p24 g p24g1 TMED1 tp24, T1/ST2 p24g2 TMED5 p28, T1/ST2 isoc p24g3 TMED7 p27 p24g4 TMED3 p26, p24b p24g5 TMED6 - d p24d1 TMED10 p23, Tmp21, p24c

* Gene names are also used to annotate proteins ** TMED, Transmembrane emp24 domain-containing protein, emp24 was the first p24 protein identified in yeast.

1.9.2 Expression An RNA expression overview of some p24 proteins in humans demonstrates that most p24 proteins are not tissue-specific (Table 1-8). In murine tissues, most members of the p24 family, for example TMED10, TMED2, TMED9, and TMED5, are expressed at similar levels in various organs, including brain, lung, liver, kidney, spleen, small intestine, colon, bladder, heart, muscles, and ovary. TMED11 is expressed in all these tissues at a much lower levels than the rest of the p24s; and only TMED6 has an high tissue specificity restricted to lung, liver, kidney, small intestine and colon (162).

Table 1-8. RNA expression of some p24 proteins in different human tissues (89)

TMED5 TMED2 TMED9 TMED2 TMED9 TMED5 TMED10 Organ Organ TMED10 amygdala 24,5 15,7 22,3 9,4 ductus deferens 47,3 41,1 56,0 10,9 basal ganglia 24,6 21,2 21,3 11,8 epididymis 52,3 49,0 46,2 12,2 cerebellum 14,0 13,2 12,4 7,6 prostate 35,6 33,3 27,3 9,1 cerebral cortex 23,0 21,5 21,5 11,1 seminal vesicle 48,9 38,0 55,7 9,2 corpus callosum 34,7 20,4 13,3 8,5 testis 29,0 17,8 20,0 7,7 hippocampal formation 25,1 15,7 17,6 9,5 breast 35,2 32,0 36,3 22,7

56 1. Introduction

TMED2 TMED9 TMED5 TMED2 TMED9 TMED5

Organ TMED10 Organ TMED10 hypothalamus 25,0 17,1 17,1 6,3 cervix, uterine 55,8 38,5 27,1 8,8 midbrain 26,9 18,2 19,1 16,5 endometrium 39,1 33,4 24,1 8,6 olfactory region 15,6 16,7 20,4 6,4 fallopian tube 38,8 27,4 22,0 8,1 pons and medulla 23,0 18,9 21,0 13,8 ovary 42,5 37,4 19,7 9,9 spinal cord 31,8 22,5 16,0 17,9 placenta 54,9 40,0 80,5 17,3 substantia nigra 26,9 18,2 19,1 16,5 vagina 30,5 27,8 28,9 19,2 thalamus 21,3 15,0 15,7 12,4 heart muscle 39,5 41,7 30,9 20,2 retina 33,8 23,8 17,5 6,0 skeletal muscle 35,7 46,3 20,5 22,7 adrenal gland 31,5 31,9 32,6 22,2 smooth muscle 30,0 30,5 26,7 6,6 parathyroid gland 28,1 18,1 21,6 9,4 appendix 19,3 21,8 24,5 13,9 pituitary gland 33,8 36,3 25,5 15,6 bone marrow 5,6 23,5 20,9 21,0 thyroid gland 55,5 49,2 32,3 19,0 lymph node 42,1 29,4 38,4 11,3 esophagus 31,3 41,3 38,1 10,2 spleen 28,3 25,6 31,0 15,2 salivary gland 119,7 90,2 72,2 14,6 thymus 34,4 24,0 13,8 8,4 tongue 27,8 23,8 24,1 8,4 tonsil 39,1 30,0 28,2 10,2 colon 28,2 31,6 29,5 11,4 B-cells 15,6 32,4 32,3 14,8 duodenum 22,9 27,6 21,0 12,8 dendritic cells 27,3 69,3 96,6 15,9 rectum 31,4 32,8 31,3 10,8 granulocytes 31,9 58,9 35,3 30,2 small intestine 31,0 34,2 21,2 12,5 monocytes 17,8 40,9 47,6 20,2 stomach 28,7 32,4 31,3 11,4 NK-cells 37,5 39,5 33,6 8,9 gallbladder 30,5 28,9 28,4 12,1 T-cells 37,9 61,2 44,0 16,8 liver 70,3 120,1 91,8 57,4 total PBMC 30,9 36,2 51,5 15,3 kidney 37,4 32,6 37,4 30,4 lung 28,1 28,1 29,9 15,8 urinary bladder 29,6 34,0 22,0 12,5 pancreas 125,6 135,4 134,5 13,6 adipose tissue 34,2 31,3 23,3 16,5 skin 26,0 27,4 24,0 6,2

1.9.3 Structure p24 proteins are type I transmembrane proteins with a molecular weight of about 24 kDa. They consist of an N-terminal, beta strand-rich globular domain called GOLD (Golgi dynamics) that facilitates protein-protein interactions; the a-helical coiled-coil domain; the transmembrane domain; and a short cytosolic tail (13-20 amino acids) that contains signals for binding to COPI and COPII vesicles. The cytoplasmic tails of p23 proteins, with an exception of the g-subfamily, are well conserved, and contain the sorting motifs like the FF-motif necessary for the ER-export, and an ER-retrieval KKXX motif (163).

57 1. Introduction

1.9.4 Cellular localization p24 proteins are found mainly in the ER, ERGIC, and Golgi, and they are enriched in COPI and COPII vesicles. As they are involved in vesicular transport, p24 proteins circulate between the compartments of the early secretory pathway (164, 165). Some fractions of p24 proteins were also found in peroxisomes, secretory granules, and the plasma membrane (163).

1.9.5 Function p24 proteins are involved in the formation of COP vesicles; cargo selection during vesicular transport, specifically selection of GPI-anchored proteins, Wnt proteins that control animal development, G-protein-coupled receptors (GPCRs) and TLRs; and secretory pathway quality control and membrane organization (163, 166). Deletion of p24 protein TMED10 in mammals is lethal, suggesting a role in embryonic development (167). In contrast, in yeast p24 proteins are not essential for vesicular transport, and their knockdown does not impair cellular growth (168).

The a-helical coiled-coil domains of p24 proteins enable their oligomerization and recognition of GPI-anchored proteins. Hetero-oligomerization of p24 proteins regulates their properties, and it has been suggested that depending on the oligomeric states p24 proteins have different functions and trafficking, localization, and stability phenotypes. Very often, deletion of one oligomeric p24 component results in downregulation of the partner component (165, 169–171). Literature reports on the function and cellular location of specific p24 proteins are often contradicting and ambiguous, which may be partially explained by the fact that p24 proteins have multiple roles depending on their oligomerization status (163).

1.9.6 TMED10 and other p24 proteins that interact with gp40 As described in [1.7.1], mainly TMED10, but also TMED2, TMED5, and TMED9 form a complex with gp40, and therefore, these p24 family members will be described further.

TMED10 forms a complex with TMED2 in mammalian cells, and this hetero- oligomerization is necessary and sufficient for the distribution of both components in the ERGIC and cis-Golgi network. If expressed separately, most of TMED10 or TMED2 is detectable in the ER. In the absence of TMED9 and TMED3 less of

58 1. Introduction

TMED10/TMED2 complex reaches the Golgi network. In other words, the distribution of TMED10 and TMED2 between the ER and Golgi depends on the expression ratio between all four members, TMED10, TMED2, TMED3 and TMED9 (164, 166).

The KKXX motif of TMED10 is not crucial for TMED10/TMED2 targeting to the Golgi network. However, the coiled coil domains of both proteins are necessary for the proper sorting, and deletion of this domain from TMED10 or TMED2 results in accumulation of both components in the ER. Importantly, when one of the complex components is missing, the cellular level of the other is reduced (166, 171).

Deletion of TMED10 in mice is lethal, however, heterozygous TMED10+/- mice are viable and fertile, and of normal size. Partial knock-down disrupts the morphology of the Golgi membranes and stability and cellular distribution of TMED3 and TMED9 (167). The half-life of TMED10 is about three hours, and it is degraded by the ubiquitin-proteasome pathway. Upon inhibition of the proteasome, TMED10 accumulates in the Golgi (172).

As generally reported for the p24 family, TMED10 together with TMED2 is required for the maturation of GPI-anchored proteins, for example decay-accelerating factor (DAF, also called CD55) or CD59, both involved in the regulation of the complement system. Deletion of TMED10 results in impaired transport of those proteins (169,170).

Even though TMED10 is localized in the early secretory pathway, especially in the cis-Golgi, literature reports demonstrate that small fraction of TMED10 is also present at the cell surface (171, 173–175).

TMED10 is a component of the presenilin complex that is involved in the regulation of the g-secretase activity. Increased activity of this complex contributes to amyloid b deposition, and it is one of the hallmarks of Alzheimer’s disease. TMED10 is not essential for complex formation, but it decreases g-secretase activity. Importantly, depletion of TMED10, but not TMED2, results in the increased production of amyloid b, suggesting that TMED10 monomer, and not TMED10/TMED2 oligomer, controls the presenilin activity. The mechanism of TMED10-mediated regulation of the presenilin complex is not clear, but it is not related to the general involvement of p24 proteins in vesicular trafficking and cargo selection. Importantly, TMED10 reaches the cell surface as a part of the presenilin complex (173).

59 1. Introduction

TMED10 is highly expressed in endocrine cells, and even though it is concentrated in the cis-Golgi, some amounts are also detected at secretory granule membranes isolated from the murine pancreatic β cells (174). Some fraction of TMED10 is also present at the cell surface of mammalian SH-SY5Y bone marrow neuroblastoma cells. Systematic studies of TMED10 structure demonstrated that the lumenal domain is not necessary for incorporation into COPI vesicles, and it promotes the membrane trafficking phenotype, whereas the cytoplasmic KKXX motif regulates cycling in the early secretory pathway (175).

Additionally, TMED10 is necessary for the transport of the misfolded GPI-anchored PrP*, a prion protein that is involved in the development of neurodegenerative disorders. During ER stress, majority of PrP* traffics from the ER to the lysosome as a complex with ER chaperones, like calnexin or BiP (Binding immunoglobulin protein), and p24 proteins, mainly TMED10 and TMED2. Importantly, all of the complex components are present at the cell surface, and deletion of TMED10 or TMED2 impairs the trafficking and lysosomal degradation of PrP*. Deletions of TMED5 and TMED9 do not inhibit, but slow down the process, suggesting their indirect involvement. These findings demonstrate that TMED10 is involved in the trafficking of misfolded GPI-anchored proteins, and that underway it reaches the cell surface (171).

Multiplexed proteomic analysis show that TMED10, as well as TMED9, but not TMED2 or TMED5, are upregulated at the cell surface during HCMV infection, even though the total cellular levels of those proteins are unchanged. This demonstrates that during infection, proteins that localize mostly in the early secretary pathway are relocated, perhaps due to the rearrangement of the cellular membranes [1.1.7] (Figure 7). Inhibition of proteasomal or lysosomal pathways does not influence protein levels of those p24 members, however viral block deletion screens suggest that the HCMV immunoevasins US1-US11 may impact the levels of p24 proteins (176).

60 1. Introduction

Plasma Whole cell Plasma Whole lysate membrane lysate

TMED10 TMED2

TMED5 TMED9 Relative abundance Relative

Time/hours

Figure 1-7. Enrichment of p24 proteins at the cell surface and in the whole cell lysate during HCMV infection (176).

Taken together, these findings indicate that the retention/retrieval signals can overbalanced by an unknown mechanism, and that TMED10 may be involved in the secretory processes in the endocrine cells and during ER stress or viral infection.

1.10 Open questions and aims of the project The previously described fundamental knowledge on the gp40 effect on RAE-1g has led to more specific questions about their interaction [1.5, 1.7]. Moreover, since some of the previously published data were not independently confirmed by others, I decided to broaden the scope of the study in order to confirm those findings.

In my project proposal I listed groundwork questions that helped to form specific aims and hypotheses:

1. Do gp40 and RAE-1g bind to each other in the cell?

61 1. Introduction

2. Does gp40 influence only the transport or also the folding and maturation of RAE-1g?

3. Does gp40 retain RAE-1g inside the cell, or does it trigger its degradation?

4. Does the interaction between gp40 and RAE-1g depend on some other molecular factor, for example a mutual binding partner?

5. How is the interaction between gp40 and class I different from the interaction of gp40 with RAE-1g?

6. Does the interaction between gp40 and RAE-1g depend on the type of RAE-1g anchorage in the membrane?

7. Does gp40 interact with class I and RAE-1g at the same time, or during different stages of viral infection?

8. Which interaction is tighter: that between gp40 and class I, or that between gp40 and RAE-1g?

9. Is the interaction between gp40 and RAE-1g structurally analogous to the interaction between human ULBP proteins and HCMV UL16?

The aims at the beginning of the project were:

1. To establish how RAE-1g traffics when it is expressed alone, or co-expressed with gp40

2. To learn about the mechanism of RAE-1g cell surface downregulation

3. To find if there is an interaction partner of gp40 that is necessary for RAE-1g downregulation, and to identify such a protein.

4. To validate our findings created with ectopically expressed proteins in cells infected with MCMV.

5. To investigate if cell membrane anchorage of RAE-1g plays a role in the interaction with gp40.

Aims 1-3, and partially aim 4 were successfully achieved, and the results are presented in the manuscript entitled “The MCMV immunoevasin gp40/m152 inhibits NKG2D receptor activation by intracellular retention and cell surface masking of

62 1. Introduction

RAE-1g” [4]. Aims 4 and 5 were not achieved due to time restrictions of the project, and therefore require further investigation.

63 2. Materials and methods

2 Materials and methods

2.1 Materials All materials are described in the Materials and Methods section of the submitted manuscript [4]. All cell lines are described in the results section of the submitted manuscript [4].

2.1.1 Constructs The original gp40 constructs were received from Venkat Raman Ramnarayan, and the details of the constructs can be found in his PhD thesis (Chapter 2.1) (177). Cloning procedures were performed in the same way as described in the PhD thesis of Linda Janßen (Chapter 2.1) (178).

Primers used to generate gp40 mutants: E28A, D113A, D236A, untagged gp40 and myc-tagged gp40 are described in the Table 2-1.

Table 2-1. Primers used for cloning of gp40 mutants and myc-tagged gp40

Mutation Primer ID Orientation 5’ to 3’ sequence

gp40 E28A oLis 4 F Forward GCGACGTTCATCTCGTACAGAGCC GGCCCGGAGATGTTCCGCCACAG oLis 4 R Reverse CTGTGGCGGAACATCTCCGGGCCG GCTCTGTACGAGATGAACGTCGC gp40 D113A oLis 5 F Forward GTACCGTACTTCAGATGATGGCTG GCAATACGTTGACACGTTAC oLis 5 R Reverse GTAACGTGTCAACGTATTGCCAGC CATCATCTGAAGTACGGTAC gp40 D236A oLis 6 F Forward GAAGGTGGAAGCGTGGCTCGCTCT CAGAGACCTGAACGGTAG oLis 6 R Reverse CTACCGTTCAGGTCTCTGAGAGCG AGCCACGCTTCCACCTTC Removing HA tag from oRVR 34 F Forward GTATCGCCTGGTCTACATCAACTG gp40 AGGATCCCCATCTTTCTAG oRVR 35 R Reverse CTAGAAAGATGGGGATCCTCAGTT GATGTAGACCAGGCGATAC Adding N-terminal myc tag Lis 7F gp40 Forward GGCGAGACGGCGGGCAGCGGAGGA to gp40 myc C GAACAAAAACTCATCTCAGAAGAG GATCTGGGAGGAAGCTATATGGAC GTG

64 2. Materials and methods

Mutation Primer ID Orientation 5’ to 3’ sequence

Lis 7R gp40 Reverse CACGTCCATATAGCTTCCTCCCAG myc C ATCCTCTTCTGAGATGAGTTTTTG TTCTCCTCCGCTGCCCGCCGTCTC GCC Adding C-terminal myc tag Lis 8F gp40 Forward CGCCTGGTCTACATCAACGAACAA to gp40 myc N AAACTCATCTCAGAAGAGGATCTG TGAGGATCCCCATCTTTC Lis 8R gp40 Reverse GAAAGATGGGGATCCTCACAGATC myc N CTCTTCTGAGATGAGTTTTTGTTC GTTGATGTAGACCAGGCG

Primers used to make HA-RAE-1g mutants, soluble HA-RAE-1g, and the HA-RAE-1g/H-2Kb chimera are described in the Table 2-2.

Table 2-2. Primers used for cloning of RAE-1g mutants

Mutation Primer ID Orientation 5’ to 3’ sequence

Soluble HA-RAE-1g Lis F HA Forward CTTCCACCTCCCGGTCACGGTACT RAE1g no GTTAAGAAAGGATTTATCG GPI_F Lis F HA Reverse CGATAAATCCTTTCTTACCAGTAC RAE1g no CGTGACCGGGAGGTGGAAG GPI_R HA-RAE-1g/H-2Kb Lis G1.1 Forward ACCCTCATTCTCTAGCCTAGGTGG chimera pJET HA GACTCATCTTCAAATCTGAGG RAE1g_F Lis G1.2 Reverse GGGAGGTGGAAGTGGGGAAG pJET HA RAE1g_R Lis G1.3 Forward CTTCCCCACTTCCACCTCCCGGTG H2Kb in GCGGAAACATGGCGACCGTTGCTG IP_F Lis G1.4 Reverse CTAGGCTAGAGAATGAGGGTCATG H2Kb in IP_R

65 2. Materials and methods

2.2 Methods

2.2.1 Cell culture, NK cell isolation, cytotoxicity assays Cell culture, NK cell isolation and cytotoxicity assays are described in the Materials and Methods section of the submitted manuscript [Error! Reference source not found.].

2.2.2 Lentiviral transduction Production of the viral particles and transduction of cells are described in [2.3.2] and [2.3.3].

2.2.3 Immunofluorescence microscopy Immunofluorescence microscopy was performed by Dr. Zeynep Hein and it is described in the Materials and Methods section of the submitted manuscript [Error! Reference source not found.].

2.2.4 Radioactive labeling and immunoprecipitation Pulse chase protocol used in this thesis is based on the pulse chase protocol published in Fritzsche & Springer (179).

The day before the experiment was performed, the cells were transfected and protein A beads were mixed with the hybridoma supernatant; 20 µl of beads slurry with 100 µl of hybridoma supernatant per each sample/time point; and incubated overnight, at 4 oC, rotating. On the day of the experiment cells were collected and resuspend cells in 3 ml of pre-warmed labeling medium, and incubate for 40 minutes o at 37 C, 5% CO2. Next, cells were resuspended in 1 ml of fresh pre-warmed labeling medium. Cells were labeled using 2-5 µl of Express Protein Labeling Mix [35S] per sample (pulse). The labeling potency of labeling mix decreases with time, therefore the volume should also be increased if the labeling stock is stored for more than

o 2 months. After incubation for 10 minutes at 37 C, 5% CO2, the pulse was finished by adding pre-warmed compete cell culture media to quench the labeling reaction. The volume of pre-warmed compete cell culture media and volume of sample was adjusted based on the number of samples/time points. Cells were incubated at 37 oC,

5% CO2 and the samples were collected as planned chase time points. Collected samples were stored on ice. After the end of the chase, cells were pelleted at 700 x g

66 2. Materials and methods for 5 minutes and the pellets were lysed using 500 µl of cold lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 supplemented with protease inhibitors (5 mM iodoacetic acid (IAA), 1 mM phenylmethylsulfonylfluoride (PMSF)) per sample. After resuspension, samples were incubated at 4 oC for 40-45 minutes, rotating. Protein A beads preincubated with hybridoma supernatant were pre-washed using cold wash buffer and distributed between the desired number of centrifuge tubes. Samples were pelleted at 16 000 x g for 10 minutes. The supernatants were carefully transfer the into the protein A beads preincubated with hybridoma supernatant and incubated for 1-2 hours, at 4 oC, rotating. Afterwards, the beads were washed three times in cold wash buffer and dried by aspiration with a syringe.

2.2.5 Co-immunoprecipitation and re-immunoprecipitation, EndoF1 digest, PNGase digest, SDS-PAGE Co-immunoprecipitation and re-immunoprecipitation, EndoF1 digest, PNGase digest, SDS-PAGE, and autoradiographic analysis were performed in the same way as described in the PhD thesis of Dr. Linda Janßen (Chapter 2.6) (178).

2.2.6 Immunostaining and flow cytometry Immunostaining and flow cytometry were described in [2.3.1]. Total volume of antibody dilution was 100 µl. Antibody and hybridoma supernatant dilutions are described in the Table 2-3.

Table 2-3. Antibody dilutions used for cell staining and flow cytometry

Antibody Dilution

gp40 1:100 m123 1:100 CX1 1:500 12CA5 1:4 Y3 1:1 Secondary antibody (APC) 1:400

2.2.7 MCMV infection Cells were seeded in a 6-well plate 24 hours before infection. Next, cells were infected with a multiplicity of infection (MOI) of 0.5. The virus was diluted in the pre-warmed,

67 2. Materials and methods complete cell culture media, and a total volume of 1 ml per well was used. The infection was performed using centrifugal enhancement of 2000 rpm for 15 minutes repeated twice (after the first spin, the 6-well plate was rotated in the centrifuge slot). o Cells were incubated at 37 C, 5% CO2, and the media was replaced with fresh, pre-warmed complete cell culture media after 4 hours or after 24 hours. Cells were incubated for the desired amount of time. Then, cells were collected and pretreated with the FcR blocking reagent (Miltenyi Biotec) diluted 1:10, for 15 minutes at 4 oC. Samples intended for intracellular staining were fixed with 2.5% PFA for 20 minutes at 4 oC and permeabilized with 0.1% Triton X-100 for 15 minutes at 4 oC. Next, samples were stained as described in [2.3.1].

2.3 Standard operating procedures As a part of the methods description, three standard operating procedures (SOP) were established. The following SOPs explain how to perform flow cytometry of mammalian cells (NL 01), production of lentiviruses (NL 02), and lentiviral transduction of cells (NL 03).

2.3.1 SOP: Flow cytometry of mammalian cells

Springer Group Standard Operating Procedure (SOP)

SOP No.: NL 01

Title: Flow cytometry of mammalian cells

Revision No.: 2

Revision Date: 2020.07.07

1. Information about this Standard Operating Procedure

SOP No., Title, Revision No., Revision Date: see page header

Author of this Revision: Natalia Lis

Signature of Principal Investigator:

68 2. Materials and methods

1. Information about this Standard Operating Procedure

Revision Author Date No. Revision History: 1 Natalia Lis 2020.06.22

2 Pawel Andruszkiewicz 2020.07.07

Other SOPs, documents, or attachments required for the procedure

2. Purpose and general description of the procedure (1-2 sentences)

Flow cytometry of mammalian cells. The SOP explains how to stain mammalian cells with antibodies or hybridoma supernatants, and how to use the flow cytometer.

3. Terms and abbreviations used in this document

Term or Explanation Abbreviation

SOP Standard Operating Procedure

PBS Phosphate-buffered saline

FBS Fetal bovine serum

ddH2O Double distilled water

RT Room temperature

4. Cells or plasmids required

- Cells that express proteins that are going to be tested - Control cells (cells transfected with empty vector as a control and/or untransfected cells) (check section 12, Instructions for the use of SOPs)

69 2. Materials and methods

5. Chemicals required

Chemical Company and Catalog Safety?4 Batch?5 No.

6. Safety considerations

6

Chemical or rcinogenic Safety considerations (H/P numbers, S1/S2, Reagent Radioactivity) Toxic Ca Allergen

Cleaning + H319 Causes serious eye irritation. solution

7. Buffers and stock solutions (='reagents')

Reagent Preparation, aliquoting, storage

PBS For 1 L of 10 x PBS dissolve 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4, 1 L ddH2O. Dilute to 1 x with ddH2O .

Cold PBS Store PBS at 4 oC

Antibodies Store as recommended by the manufacturer. Antibodies purified from hybridoma supernatant are kept in 4 °C.

Hybridoma Hybridoma supernatants (unpurified) are kept at 4 °C, and supernatants their stock solutions are kept at -20°C.

Cell culture media Prepare complete cell culture media as recommended for the type of cells used in the experiment. Usually it is DMEM for adherent cells and RPMI for suspension cells, and it is supplemented with 10% of FBS, 1% penicillin/ streptomycin (Pen/Strep mix contains 10,000 units potassium penicillin and 10,000 μg streptomycin sulfate per mL) and 1% L-Glutamine (200 mM).

4 Insert the 'Signal word' (Warning, Danger, …) of the GHS system (in the Lab chemicals database) if it applies. And enter detailed safety information in table 6 (Safety). 5 Insert 'Yes' if necessary to record the batch number of this chemical. 6 Check whatever applies.

70 2. Materials and methods

7. Buffers and stock solutions (='reagents')

Reagent Preparation, aliquoting, storage

Trypsin Storage 4 °C

Sheath fluid Storage RT

Cleaning solution Storage RT

Decontaminating Storage RT solution

8. Equipment and accessories required

Type of equipment Special Instruction?7

Cell culture bench Yes

Cell culture 6-well plates

96-well plates V-bottom clear wells

Pump

Micropipettes (2, 20, 200, 1000 μl)

Micropipette tips (2, 20, 200, 1000 μl)

Multichannel pipette (8 or 16 channels)

Microcentrifuge tubes 1.5 ml and 2 ml

Flow cytometer Yes

Flow cytometry tubes

Ice

9. Procedure (numbered list)

If you work with adherent cells, start with step 1 of the procedure. If you work with suspension cells, start with the step 7 of the procedure. 1. Warm up trypsin to 37 oC. 2. Wash cells with 1 x PBS, 2 ml per well.

7Insert 'Yes' if special instruction is necessary to operate this equipment.

71 2. Materials and methods

9. Procedure (numbered list)

3. Add 250 µl of trypsin per well, incubate for 2-3 minutes at 37 oC. Check if cells are detaching from the plate surface. You may tap the side of the 6-well plate gently to check if the cells are detaching. 4. Add 1 ml cell culture media. 5. Collect cells the into the microcentrifuge tube. 6. Optional: wash the well with 500 μl of PBS to collect the residual cells, and add them to already collected cells. 7. Spin down the cells at 250 x g for 3 minutes Remove the supernatant (using pipette or the vacuum pump). Resuspend cells in 200 ml of cold PBS 8. Transfer cells to the V-shape 96-well plate. From now on keep the plate on ice, use cold PBS, and store all reagents on ice. 9. Spin down the cells at 250 x g for 3 minutes Remove the supernatant. 10. Resuspend cells in primary antibody solution or hybridoma supernatant. 11. Incubate on ice for 45 minutes. Protect your samples from light if antibodies are conjugated to fluorophores. 12. Fill up the volume in the well up to 200 μl. 13. Spin down the cells at 250 x g for 3 minutes Remove the supernatant. Resuspend cells in 200 ml of cold PBS (this is the washing step). 14. Spin down the cells at 250 x g for 3 minutes Remove the supernatant. 15. Resuspend cells in secondary antibody solution. 16. Incubate on ice for 45 minutes. Protect your samples from light if antibodies are conjugated to fluorophores. 17. Fill up the volume in the well up to 200 μl. 18. Spin down the cells at 250 x g for 3 minutes Remove the supernatant. Resuspend cells in 200 ml of cold PBS. Repeat two times (two washes with cold PBS). 19. Spin down the cells at 250 x g. Remove the supernatant. Resuspend cells in 200 μl of cold PBS. 20. Transfer cells to the flow cytometry tubes. Increase the sample volume in the tube up to 500 μl if you use 1.2 ml flow cytometry tube, or 1.2 ml if you use 3.5 ml flow cytometry tube. Store your samples on ice and protected from the light. 21. IMPORTANT: Check the containers connected to the flow cytometer. The waste container should not be full. If it contains more than 500 ml of waste, empty it (S1 waste) by transferring the waste to another bottle. Do not exchange the waste bottle connected to the flow cytometer as it is high vacuum-resistant. The sheath fluid container should not be empty. If it is less than 300 ml, refill it up to the level indicated on the bottle. Make sure that both containers ale closed tightly. Check the levels of liquid in both containers while running the samples. 22. Turn on flow cytometer. 23. Turn on flow cytometer software FloMax 3.0 and CCD-Camera. 24. Choose cleaning settings. If you plan to use the red laser, choose cleaning with the red laser. Clean the machine two times. Make sure that there is at least 2 ml of cleaning solution in the 3.5 ml flow cytometry tube used to clean the instrument. 25. Choose page layout, and settings.

72 2. Materials and methods

9. Procedure (numbered list)

26. Run your samples (vortex shortly before each measurement). 27. Choose cleaning settings. Clean the machine two times. Make sure that there is at least 2 ml of cleaning solution in the 3.5 ml tube. 28. Choose decontamination settings. Decontaminate the machine one time. Make sure that there is at least 1 ml of decontaminating solution in the tube. 29. Run the sheath fluid using the decontamination settings, or increase the speed up to 10 μl/s, run the sheath fluid, and manually stop after 1 minute. 30. Turn off the flow cytometry software. 31. Turn off the flow cytometer. 32. Exchange the tube containing the sheath fluid to an empty tube. 33. Empty the waste container and refill the sheath fluid if necessary (look step 22). 34. Sign in to the log book. 35. Discard leftover samples (S1 waste). Discard tubes used for cleaning and decontamination.

10. Interpretation and reporting of the data:

Transfer all the data to the server folder. Every sample name should contain type of cells, type of staining, and experiment number and date of experiment. Use FloJo software to create the data report. Use online tutorial to learn about all the options and methods for flow cytometry data analysis and presentation (https://www.flowjo.com/learn/flowjo-university/flowjo). Read published guides for the flow cytometry data interpretation, for example: L A Herzenberg et al. Interpreting flow cytometry data: a guide for the perplexed. 2006. Nature Immunology volume 7, pages681–685(2006). More useful flow cytometry guides are also available in the Server Folder/People/Natalia/SOP.

11. Potential pitfalls, errors, and other issues (each in one table row)

Issue Known resolution

No cells in the sample. Make sure that after each spinning down of the cells there is a pellet. Small pellet may be visible only before removing of the supernatant. If there is no pellet, increase the time of spinning to 5 minutes. Some types of cells are more sensitive to spinning and pipetting. You should resuspend them gently and use pipette tips with rounded end. You may also decrease the speed to 200 x g and increase the time to 5-6 minutes.

73 2. Materials and methods

11. Potential pitfalls, errors, and other issues (each in one table row)

Issue Known resolution It usually helps to keep the sample volume high while spinning down and removing the supernatant after the spin. Therefore, fill up the well volume up to 200 μl before spinning.

Cross contamination between Avoid spill over when resuspending your cells. samples. Always include the same types of controls between the experiment repeats. Change tips between each step.

Antibody staining does not work. Optimize antibody dilution. The antibody dilution is best optimized in respect to constant cell count. Include positive antibody controls. Check if antibodies are stored according to the manufacturer recommendation. Use fresh vial of the antibody. Increase staining time up to 60 minutes.

Hybridoma staining does not work. Test your stock for bacterial/fungi contamination. Test your stock for cross-contamination with other hybridomas or reagents. Check if the hybridoma stock stored at 4 oC is old (open for more than 1 year) and used by many people. Use fresh aliquot. Increase staining time up to 60 minutes.

Unspecific signals in some Resuspend the pellets well while washing with samples. PBS. Increase number of washing steps after every staining, for example wash 2-3 times after each staining step.

Contamination of the flow Wash the flow cytometer with cleaning solution cytometer tubing. and decontamination solution multiple times, Unspecific signals when running until the contamination is no longer detectable. your sample. Ask for technical assistance.

74 2. Materials and methods

12. Instructions for the use of SOPs

General Rules: • Good starting number of cells for one staining sample (one flow cytometry sample) is 1x106. An experienced user may go down to ~2.5-3x105 cells/sample. • This protocol is written for staining of cells seeded in the 6 well plate. You can down/up-scale the number of cells using different cell culture dishes. Adjust volumes of solutions used for collection of the cells. The staining steps remain the same. • Plan what kinds of controls you will need. Cell controls: unstained cells for adjustment of the machine settings; cells transfected with an empty vector, stained with the same combination of antibodies as your samples Antibody controls: secondary antibody, single antibody (if you do multiple staining of your sample), isotype-matched unspecific antibody. • Optimize dilution of antibodies used for staining. Usually there is a dilution range recommended by the manufacturer. If not, start with dilutions 1:50, 1:100, 1:200, 1:500. Optimize dilution of the hybridoma supernatant used for staining. Start with dilutions 1:1, 1:3, 1:20. Always use cold PBS to make dilutions and store all solutions on ice. • When you use an antibody conjugated with a fluorophore, protect the solution form light. While staining cells with fluorophore-conjugated antibody, protect your samples from light. • If you use fluorophores that have overlapping emission spectra, use controls to compensate the unspecific detected signals. Compare excitation and emission spectra of fluorophores when planning the experiment with multiple fluorophores. • Good staring settings to run your samples are: Sample speed: 1 μl/s (1.2 ml tube) or 3 μl/s (3.5 ml tube). FSC gain 120, log3. SSC gain 200 log3. FL1 gain 315, log4. FL2 gain 320, log4. FL5 gain 615, log4. • Use the newest version of an SOP for your experiment. • Record the number of the SOP (found in the page header) in your experiment protocol. • Any changes between the SOP and your experiment must be documented in your experiment protocol. • If you believe that the SOP needs to be changed or extended, bring it up in the subgroup meeting. Explanations of the individual points: 3. Abbreviations: Abbreviations of chemicals are explained in 5. 5. "Chemicals" are all powders (but not stock solutions, see 6.)

75 2. Materials and methods

2.3.2 SOP: Production of lentiviruses

Springer Group Standard Operating Procedure (SOP)

SOP No.: NL 02

Title: Production of lentiviruses

Revision No.: 1

Revision Date: 2

1. Information about this Standard Operating Procedure

SOP No., Title, Revision No., Revision Date: see page header

Author of this Revision: Natalia Lis

Signature of Principal Investigator:

Revision Author Date No. Revision History: 1 Natalia Lis 2020.06.22

2 Pawel Andruszkiewicz 2020.07.07

Other SOPs, documents, or attachments required for the Cell culture training procedure

2. Purpose and general description of the procedure (1-2 sentences)

Production of viral particles that may be used for transduction of mammalian cells.

3. Terms and abbreviations used in this document

Term or Explanation Abbreviation

SOP Standard Operating Procedure

PEI Polyethylenimine

76 2. Materials and methods

3. Terms and abbreviations used in this document

Term or Explanation Abbreviation

FBS Fetal Bovine Serum

PBS Phosphate-buffered saline

ddH2O Double distilled water

RT Room temperature

4. Cells or plasmids required

HEK293T cells

For expression of a protein: Packing vector NLBH (Strain collection number 1505) Envelope vector VSV-G (Strain collection number 980) Transfer vector (vector that encodes construct to be expressed in mammalian cells)

For a knock-out of a protein: Packing vector psPAX2 (Strain collection number 1455) Envelope vector VSV-G (Strain collection number 1457) Transfer vector lentiCRISPR v2 (Strain collection number 1454). Clone the guide RNA intro this vector.

5. Chemicals required

Chemical Company and Catalog Safety?8 Batch?9 No.

8 Insert the 'Signal word' (Warning, Danger, …) of the GHS system (in the Lab chemicals database) if it applies. And enter detailed safety information in table 6 (Safety). 9 Insert 'Yes' if necessary to record the batch number of this chemical.

77 2. Materials and methods

6. Safety considerations

10 Chemical or Safety considerations (H/P numbers, S1/S2, Reagent Radioactivity) Toxic Carcinogenic Allergen

Bacillol + H226 Flammable liquid and vapour. H318 Causes serious eye damage. H336 May cause drowsiness or dizziness

7. Buffers and stock solutions (='reagents')

Reagent Preparation, aliquotting, storage

PEI Dissolve 200 mg of PEI in 20 ml of ddH2O to obtain a 10 x solution of PEI. Dilute to 1 x with ddH2O and filter with 0.2 mm single use syringe filter. Store aliquots at -20oC. After thawing, store at 4 oC.

Cell culture media Prepare complete cell culture media as recommended for the type of cells used in the experiment. Usually it is DMEM for adherent cells and RPMI for suspension cells, and it is supplemented with 10% of FBS, 1% penicillin/streptomycin (Pen/Strep contains 10,000 units potassium penicillin and 10,000 μg streptomycin sulfate per mL) and 1% L-Glutamine (200 mM).

PBS Storage RT

Trypsin Storage 4oC

Bacillol Storage RT

8. Equipment and accessories required

Type of equipment Special Instruction?11

Cell culture bench Yes

10 Check whatever applies. 11Insert 'Yes' if special instruction is necessary to operate this equipment.

78 2. Materials and methods

8. Equipment and accessories required

Type of equipment Special Instruction?11

10 cm cell culture dish

15 mL centrifuge tubes

Single use syringe filter

Single use sterile syringe

Disposable laboratory gloves

Lab coat

Serological Pipettes

Pipetboy

Micropipettes (2, 20, 200, 1000 μl)

Micropipette tips (2, 20, 200, 1000 μl)

Microcentrifuge tubes 2 mL

9. Procedure (numbered list)

All steps should be done in the clean cell culture bench. All steps should be done while wearing clean lab coat and disposable laboratory gloves (standard cell culture procedures). Steps 1-7 may be done in S1 cell culture bench. Steps 8-14 must be done in the S2 cell culture bench. Alternatively, all steps may be done in the S2 cell culture bench. Keep separate aliquots of the complete cell culture media, trypsin and PBS in the S2 laboratory, and do not use it in the S1 laboratory. 1. Seed HEK293T cells in the 10 cm cell culture dish. The confluency on the day of transfection should be 60-70%. This step may be done 1-2 days (recommended) or few hours before the cell transfection. The cells should have enough time to adhere to the cell culture dish. 2. On the day of transfection remove the supernatant from the plate with HEK293T cells. Add 8 ml of pre-warmed cell culture media. 3. Mix 950 μl of transfection media with 6 mg of each plasmid: packing vector, envelope vector, transfer vector, in one 2 ml microcentrifuge tube. Mix vigorously. 4. Mix 950 μl of transfection media with 45 μl of PEI in one 2 ml microcentrifuge tube. Mix vigorously. 5. Incubate both mixtures for 30 minutes in room temperature. 6. After 30 minutes of incubation mix content of tubes prepared in steps 4 and 5. Mix gently.

79 2. Materials and methods

9. Procedure (numbered list)

From this step onwards, work in the S2 cell culture bench 7. Add mixture prepared in step 6 to the HEK293T cells (step 6). 8. Incubate cells for 20-22 hours. 9. Remove the supernatant from the plate with HEK293T cells. Add 8 ml of pre-warmed cell culture media. 10. Incubate cells for 24 hours. 11. Collect the supernatant into the 15 ml centrifugal tube. Discard the cell culture plate. 12. Filter the supernatant using 0.45 μm single use syringe filter. - Open 15 ml centrifugal tube and place it in a steady position. - Place the filter on the top of the tube - Take out the syringe plunger - Place the syringe hub into the filter hub - Pour the virus-containing supernatant into the syringe barrel - Put back the syringe plunger into the barrel - Press the plunger while holding the filter and tube tightly. The filtered supernatant will be collected in the new 15 ml tube. 13. Store the supernatant that contains the filtered virus in 4 oC (weeks) or -20 oC (moths).

10. Interpretation and reporting of the data:

Check SOP Lentiviral transduction of cells NL 03.

11. Potential pitfalls, errors, and other issues (each in one table row)

Issue Known resolution

Cells die Check for contaminations in the cell culture media, trypsin and PBS. Use fresh ones in necessary. Check if the cell culture incubator works properly. Decrease volume of PEI down to 40 µl. Use fresh DNA preps.

Viral transduction does Check the quality of the DNA preps. Use fresh ones if not work necessary. Use fresh stock of HEK293T cells for the production of viruses. HEK293T cells should be passaged at least two times before using them for the virus production. Make sure that the target cells that you are transducing are compatible with the lentiviral procedure.

80 2. Materials and methods

12. Instructions for the use of SOPs

General Rules: • As transfection media use incomplete cell culture media (without FBS, penicillin/streptomycin and glutamine) or commercial transfection media. • For more information on the lentiviral transduction systems and methods visit:

https://www.addgene.org/guides/lentivirus/ https://blog.addgene.org/getting-the-most-from-your-lentiviral-transduction • Use the newest version of an SOP for your experiment. • Record the number of the SOP (found in the page header) in your experiment protocol. • Any changes between the SOP and your experiment must be documented in your experiment protocol. • If you believe that the SOP needs to be changed or extended, bring it up in the subgroup meeting. Explanations of the individual points: 3. Abbreviations: Abbreviations of chemicals are explained in 5. 5. "Chemicals" are all powders (but not stock solutions, see 6.)

2.3.3 SOP: Lentiviral transduction of cells

Springer Group Standard Operating Procedure (SOP)

SOP No.: NL 03

Title: Lentiviral transduction of cells

Revision No.: 1

Revision Date: 2

1. Information about this Standard Operating Procedure

SOP No., Title, Revision No., Revision Date: see page header

Author of this Revision: Natalia Lis

Signature of Principal Investigator:

81 2. Materials and methods

1. Information about this Standard Operating Procedure

Revision Author Date No. Revision History: 1 Natalia Lis 2020.06.22

2 Pawel Andruszkiewicz 2020.07.07

Other SOPs, documents, or Cell culture training attachments required for the procedure Production of lentiviruses NL 02

2. Purpose and general description of the procedure (1-2 sentences)

Transduction of mammalian cells with lentiviruses (also called retroviruses) in order to obtain cell lines that stably express the construct of interest. There are two types of mammalian expression vectors used to express proteins in the Springer laboratory. The first type of vectors contains antibiotic resistance, therefore a selection reagent (antibiotic of choice) must be used to select successfully transduced cells. The second type of vector encodes a GFP gene, therefore cells can be selected based on the expression of the GFP protein. Most common expression vectors used in Springer laboratory are: puc2CL6IPwo (Puromycin selection), puc2CL6INwo (G418/Geneticin selection) or puc2CL6IEGwo (GFP expression), abbreviated as IP, IN, and IEG.

3. Terms and abbreviations used in this document

Term or Explanation Abbreviation

SOP Standard operating procedure

PBS Phosphate-buffered saline

GFP Green fluorescent protein

RT Room temperature

4. Cells or plasmids required

Target cells (cells that will be transduced)

82 2. Materials and methods

5. Chemicals required

Chemical Company and Safety?12 Batch?13 Catalog No.

6. Safety considerations

14

Chemical or Safety considerations (H/P numbers, S1/S2, Reagent Radioactivity) Toxic nic Carcinoge Allergen

Bacillol H226 Flammable liquid and vapour. H318 Causes serious eye damage. H336 May cause drowsiness or dizziness

Selection Check for your selection reagent of choice. reagent

7. Buffers and stock solutions (='reagents')

Reagent Preparation, aliquotting, storage

Cell culture media Prepare complete cell culture media as recommended for the type of cells used in experiment. Usually it is DMEM for adherent cells and RPMI for suspension cells, and it is supplemented with 10% of FBS, 1% penicillin/streptomycin (Pen/Strep contains 10,000 units potassium penicillin and 10,000 μg streptomycin sulfate per mL) and 1% L-Glutamine. (200 mM).

PBS Storage RT

Trypsin Storage 4oC

Bacillol Storage RT

Selection reagent Check for your selection reagent of choice.

12 Insert the 'Signal word' (Warning, Danger, …) of the GHS system (in the Lab chemicals database) if it applies. And enter detailed safety information in table 6 (Safety). 13 Insert 'Yes' if necessary to record the batch number of this chemical. 14 Check whatever applies.

83 2. Materials and methods

8. Equipment and accessories required

Type of equipment Special Instruction?15

Cell culture bench Yes

12-well cell culture plate (Can be changed to 24-well cell culture plate)

16-well cell culture plate

Disposable laboratory gloves

Lab coat

Serological Pipettes

Pipetboy

Micropipettes (2, 20, 200, 1000 μl)

Micropipette tips (2, 20, 200, 1000 μl)

Microcentrifuge tubes 2 mL

9. Procedure (numbered list)

All steps should be done in the clean cell culture bench. All steps should be done while wearing clean lab coat and disposable laboratory gloves (standard cell culture procedures). All steps that include handling of the viral stocks must be done in the S2 cell culture bench. Target cells may be transferred to the S1 cell culture room at least 2 passages after the transduction step (mixing of cells with viruses). Transduction using a construct that contains antibiotic selection (puromycin of G418) A) Estimating antibiotic selection concentration: 1. Seed target cells in 10 wells of the 12-well plate. The final cell confluency should be 50%. All cells should adhere to the plate surface. 2. Add antibiotic to each well. Use 10 different concentrations, including 0 μl/ml. 3. Estimate the cell viability in each well after 24 and 48 hours. Note down the concentration that kills majority of cells after 48 hours. B) Transduction: 4. Seed small amount of target cells, 20-30 cells, into 4 wells of the 12-well plate. All cells should adhere to the plate surface.

15 Insert 'Yes' if special instruction is necessary to operate this equipment.

84 2. Materials and methods

9. Procedure (numbered list)

5. Mix cells with the viral stock (SOP NL 02 Production of viruses) at three different concentrations. For example, use 0.5/1/2 ml of the viral supernatant in the total volume of 2 ml of the pre-warmed cell culture media. Always include control cells without the virus. 6. After 24 hours exchange the supernatant with the pre-warmed cell culture media. 7. After 48 hours exchange the supernatant with the pre-warmed cell culture media and add selection reagent at the concentration established in step A3. Selection reagent should also be added to the control cells that were not mixed with the viruses. 8. After 48 hours exchange the supernatant with the pre-warmed cell culture media. 9. When cell confluency increases to about 40-50%, transfer cells to the 6-well cell culture plate and incubate for another 24 hours. 10. Test an aliquot of transduced cells for the mutation of interest. Selection of cells using a construct that contains GFP gene. 1. Seed small amount of target cells, 20-30 cells, into 4 wells of the 12-well plate. All cells should adhere to the plate surface. 2. Mix cells with the viral stock (SOP NL 02 Production of viruses) at three different concentrations. For example, use 0.5/1/2 ml of the viral supernatant in the total volume of 2 ml of the pre-warmed cell culture media. Always include control cells without the virus. 3. After 24 hours exchange the media with the pre-warmed cell culture media. 4. When cell confluency increases to about 40-50%, transfer cells to the 6-well cell culture plate and incubate for another 24 hours. 5. Test an aliquot of transduced cells for the mutation of interest.

10. Interpretation and reporting of the data:

In order to test whether the transduction worked, test your cells for expression of the transduced protein or protein knock down using flow cytometry or Western Blot, or other suitable method.

11. Potential pitfalls, errors, and other issues (each in one table row)

Issue Known resolution

The mutation of interest did not Use fresh stock of target cells for occur. transduction. Cells should be passaged at least two times before using them for the virus transduction. Prepare fresh stock of the lentiviral particles.

85 2. Materials and methods

11. Potential pitfalls, errors, and other issues (each in one table row)

Issue Known resolution Optimize the concentration of reagent selection (antibiotic kill curve). Check if your target cells may be more compatible with different kinds of transfection, like lipofectamine transfection or electroporation.

The mutation of interest occurs but Reselect the cells again using the selection the effect is not uniform between reagent. Increase the concentration of the cells. The population of selection reagent. transduced cells in not Use the cell cloning by serial dilution in homogenous. 96-well plates protocol by Corning to select single clones with desired transduction efficiency.

12. Instructions for the use of SOPs

General Rules: • In all cases it is recommended to use the cell cloning by serial dilution in 96- well plates protocol by Corning to select single clones with desired transduction efficiency

(https://www.corning.com/catalog/cls/documents/protocols/Single_cell_clon ing_protocol.pdf)

also available in the Server Folder/People/Natalia/SOP. • There are good on-line guides that describe reagent selection (antibiotic selection) curve and types of antibiotics that may be used for selection: https://www.sigmaaldrich.com/technical- documents/articles/biology/antibiotic-kill-curve.html https://www.goldbio.com/documents/1094/Titration+Kill+Curve+Protocol.pd f • Use the newest version of an SOP for your experiment. • Record the number of the SOP (found in the page header) in your experiment protocol. • Any changes between the SOP and your experiment must be documented in your experiment protocol. • If you believe that the SOP needs to be changed or extended, bring it up in the subgroup meeting. Explanations of the individual points: 3. Abbreviations: Abbreviations of chemicals are explained in 5.

86 2. Materials and methods

12. Instructions for the use of SOPs 5. "Chemicals" are all powders (but not stock solutions, see 6.)

87 3. Grant application

3 Grant application

Together with Prof. Sebastian Springer, I wrote a grant application for the Deutsche Forschungsgemeinschaft (German Research Council) entitled “Suppression of the innate immune response by cytomegalovirus: two molecular mechanisms of action of MCMV gp40/m152 on RAE-1g”.

This applications describes experimental plans regarding the gp40/RAE-1g project and another prospective future project about the interaction between the HCMV immunoevasin UL16 and human NKG2DL MICB. My role in the process was to formulate ideas, identify goals and objectives of the proposed project, plan the methodology and working strategy, and to write the initial draft.

3.1 State of the art and preliminary work

3.1.1 Scientific background of the project16

3.1.1.1 activation: NKG2D receptor and ligands Natural Killer (NK)17 cells play a crucial role in the innate immune defence against viruses. Once activated, they kill infected cells or activate other immune cells (1, 2). NK cell activation is regulated by a balance of signals from their activating and inhibiting receptors (3).

NKG2D is an activating receptor on the surface of NK cells18. It recognizes protein ligands that are expressed on the surface of other cells upon cellular stress (e.g., mutation or infection). NKG2D is also found on activated CD8+ T cells, activated macrophages, and some gd T cells. The study of NKG2D and its ligands is essential to understanding immune regulation (4, 5).

16 The section "Project-related publications" is in 3.1.3. 17 Abbreviations: CMV, cytomegalovirus; ER, ; gp40, MCMV immunoevasin that downregulates RAE-1g and MHC class I; gp40LM, gp40 linker mutant ; gp40WT, gp40 wild type; HA tag, hemagglutinin epitope tag; HA-RAE-1g, N-terminally HA-tagged RAE-1g; HCMV, human cytomegalovirus (human herpesvirus-5); HEK, human ambryonic kidney (cells); MCMV, mouse cytomegalovirus; MHC, major histocompatibility complex; MICA/B, MHC class I polypeptide-related sequence A/B; MOI, multiplicity of infection; MULT-1, Murine UL16 binding protein-like transcript; NK cell, Natural Killer cell; NKG2D, activating NK cell receptor; RAE-1 (retinoic acid-inducible 1), NKG2D ligand with 5 isoforms (RAE-1 a,b,d,g,e); siRNA, small interfering RNA; TMED, transmembrane emp24 domain-containing (the p24 protein family); ULBP, UL16-binding protein. 18 It belongs to the CD94/NKG2 family of C-type lectin-like receptors.

88 3. Grant application

All NKG2D ligands have a similar structure (with a domain organization similar to MHC (major histocompatibility complex) class I proteins), and they are recognized by NKG2D in the same fashion (6). The human NKG2D ligands are called MICA19, MICB, and ULBP1 to 6, whereas their orthologs in mouse are H60, MULT-1, and RAE-1a to e (Figure 3-1).

Figure 3-1. Mouse and human NKG2D ligands. The structure of all NKG2D ligands is similar to the structure of MHC class I molecules, but they do not bind beta-2 microglobulin (the MHC class I light chain) or peptide and (with the exception of MICA and MICB) do not contain an α3-like domain. MICA, MICB, ULBP4, MULT-1, and H60 are attached to the plasma membrane via a type 1 transmembrane domain. Other ULBPs and RAE-1 have glycosylphosphatidylinositol (GPI) anchors (red). Adapted and reprinted with permission from (6).

3.1.1.2 Cytomegalovirus evades NK cell activation 3.1.1.2.1 MCMV and HCMV Cytomegalovirus (CMV) is a frequent and severe threat to immunocompromised individuals (such as organ transplant recipients and cancer patients) and congenitally infected infants. Due to its broad similarity to human CMV (HCMV), murine cytomegalovirus (MCMV) is a useful model to study infection and immune evasion (7, 8). Both the HCMV and MCMV genomes encode immunoevasins, i.e., proteins that inhibit immune responses, including NK cell activation [9]. Discoveries made with MCMV have often generated hypotheses for HCMV infection, and the MCMV model allows the researcher to study the impact of viral factors in vivo (10).

3.1.1.2.2 Mechanisms of evasion from the NK cell response To block the NK cell response to infection, many CMV immunoevasins inhibit the surface expression of NKG2D ligands, usually by retaining them in the cell interior.

19 For the resolution of the name abbreviations please see footnote 17.

89 3. Grant application

For example, HCMV UL142 retains MICA (11), and HCMV UL16 retains MICB, ULBP1 and ULBP2 (12, 13). Remarkably, the molecular mechanism of this retention remains unclear in every case. Some authors have shown which domain of an immunoevasin is essential for the retention of its target NKG2D ligand20, but it remains unknown whether the immunoevasins bind directly to the NKG2D ligands, and how they achieve the retention of their targets (11, 14). In contrast, for MHC class I retention by CMV immunoevasins, one such mechanism has been elucidated by us (17, 18). We believe that understanding the molecular mechanism of the downregulation of NKG2D ligands will help with designing better strategies to fight HCMV infection.

3.1.1.3 The MCMV immunoevasin gp40 and RAE-1g downregulation The MCMV immunoevasin gp40 (m152)21 targets both the adaptive and the innate immune response. It retains antigen-presenting MHC class I molecules in the early secretory pathway (17). Krmpotić and collaborators were the first to report that gp40 also prevents NK cell-mediated lysis in vitro by targeting the NKG2D ligand RAE-122 (19, 20). The most gp40-susceptible RAE-1 isoform23 is RAE-1g (21). The group of Jonjić were the only researchers to report that gp40 retains the mature form of RAE-1g in the early secretory pathway (22).

Still, the molecular mechanism of this retention remains unknown. The following are the most prominent questions:

• Is RAE-1g downregulated by retention or also by lysosomal degradation? • Does RAE-1g bind to gp40? • What factor retains RAE-1g (or a gp40/RAE-1g complex) in the cell interior?

20 Two examples from HCMV immunoevasins: the transmembrane domain of UL142 is necessary for the retention of MICA in the ER (14), and the transmembrane and cytosolic domains of UL16 cause intracellular retention of MICB (12). 21 The names gp40 (for the protein) and m152 (for the gene) are used interchangeably in the literature. We use here the name gp40. 22 The abbreviation means 'Retinoic acid early inducible 1'. Not to be confused with the mRNA export factor RAE1 (ribonucleic acid export 1). 23 There are five isoforms of RAE-1: a, b, g, d, and e. Their sequence homology is about 90%. Their genes are called Raet1a to e. RAE-1a, b, and g are susceptible to gp40-mediated downregulation. (The genes for the human ULBP proteins are also called RAET.)

90 3. Grant application

3.1.2 Preliminary data

3.1.2.1 Interaction between gp40 and MHC class I molecules 3.1.2.1.1 gp40 retains mouse class I in the early secretory pathway Our group has closely studied the interaction between gp40 and MHC class I molecules. We and others have established that gp40 retains murine class I molecules in the early secretory pathway (i.e., the Endoplasmic Reticulum (ER) and the cis-Golgi), and that individual class I allotypes have different susceptibilities to gp40 retention (17, 23). We found that class I retention is caused by gp40 binding to class I. To identify how gp40 itself is retained in the cell interior, we mutated it and found that the linker sequence of gp40, which connects the ER-lumenal domain with the transmembrane domain, is essential for its retention. If the linker sequence is mutated to a sequence of glycines and serines, then the resulting gp40 linker mutant, gp40LM24, is no longer retained in the early secretory pathway but rapidly trafficks to the cell surface, and class I retention no longer occurs (Figure 3-2A). In contrast, gp40 with the wild type linker (gp40WT) circulates in the early secretory pathway (Figure 3-2B) and retains class I in that location (18).

3.1.2.1.2 Retention of MHC class I depends on the binding of the gp40 linker to TMED10 We next explored the function of the gp40 linker using co-immunoprecipitation followed by mass spectroscopy. We found that gp40WT (but not gp40LM) binds to the host protein, TMED10 (Figure 3-3A) (18). We established that TMED10 is essential for localizing gp40: if the TMED10 gene is knocked down or knocked out, then gp40 is no longer retained in the early secretory pathway, and surface class I is no longer downregulated (Figure 3-3B). Thus, gp40 tethers two host proteins to each other that would not normally interact (class I and TMED10) in order to retain class I inside the cell (18).

24 The mutant called here gp40LM is the gp40-GGGG mutant from (17), where the entire linker sequence was exchanged for the sequence (G4S)9.

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A H2-Kb gp40WT H2-Db gp40LM

0.0 1.0 2.0 Surface class I levels (AU)

Figure 3-2 . gp40 retains murine MHC class I proteins in the early secretory pathway. A) The gp40 linker sequence is required for the reduction of murine class I surface levels. gp40WT (wild type gp40) or gp40LM (gp40 linker mutant)-expressing murine fibroblasts were surface-stained with B22.249 (for the murine MHC class I molecule H-2Db) and Y3 (for the murine MHC class I molecule H-2Kb) and analyzed by flow cytometry. The data show that only gp40WT is able to retain H-2Db and H-2Kb inside the cell whereas gp40LM does not. The bar chart shows the mean fluorescence intensities (mean ± SEM, n = 4). B) gp40 wild type circulates in the early secretory pathway, whereas the gp40 linker mutant quickly leaves the early secretory pathway. Human HEK293T cells expressing gp40WT or gp40LM were pulse-labeled for 10 minutes, chased for the indicated times, and lysed in 1% Triton X-100 buffer. gp40 was immunoprecipitated from the lysates with an anti-HA antibody, digested with endoglycosidase F1 (EndoF1, cleaves only ER forms of glycans, like EndoH1) or peptide N-glycosidase (PNGase, cleaves all glycan forms) as indicated, and separated by SDS-PAGE. Rt, sialylated band; S, EndoF1-sensitive band; U, undigested bands (gp40 has two glycan isoforms that run as separate bands); black arrows, partially EndoF1-resistant bands; asterisk, unspecific band. Work of the Springer group, adapted from (18).

A B siRNA

Untreated Tmed10

gp40WT gp40LM 0.1 99.6 0.2 99.4

WB: gp40 (HA) - gp40 IP: gp40 (HA) 0.05 0.19 0.07 0.28 WB: TMED10 Untreated Tmed10 98.8 0.89 42.3 55.7

urface CD29 levels CD29 urface + gp40 S

0.29 0.0 0.93 0.09

Surface class I levels

Figure 3-3. Intracellular retention of class I depends on the binding of the gp40 linker to TMED10. A) The gp40 linker sequence binds to TMED10. K41 murine fibroblasts expressing gp40WT or gp40LM were lysed in 1% digitonin buffer. gp40 was immunoprecipitated using an anti-HA antibody, and the proteins were separated by SDS-PAGE and immunoblotted for gp40 and TMED10. It shows that gp40WT binds to TMED10 while gp40LM does not. B) gp40 uses TMED10 to retain MHC class I inside the cell. K41 cells expressing either the empty vector (- gp40) or gp40WT (+ gp40) were transfected with 300 pmol of siRNA against TMED10. After 60 hours, the cells were harvested and co-stained for cell surface class I and CD29 as a control. The cells were analyzed by flow cytometry, and the data are shown as two-dimensional plots of cell surface class I levels (x axis) and CD29 (y axis). It turns out that the loss of TMED 10 restores cell surface class I levels in the presence of gp40. One representative experiment out of six is shown. Work of the Springer group, adapted from (18).

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3.1.2.1.3 Background on the p24 family TMED10 is a member of the p24 family of proteins that circulate between the ER and the Golgi apparatus25. The p24 proteins are highly conserved among all eukaryotes, and there are ten family members in mammals. All p24 proteins are type 1 single-pass transmembrane domains of 20-25 kDa with a large lumenal domain, and some (among them TMED10) carry the cytosolic C-terminal ER retention motif, –KKXX (24). They form heterodimers and -tetramers and are all localized to the ER and the cis-Golgi26, with some especially enriched in the membranes of the COPII (ER-to-Golgi) and the COPI (Golgi-to-ER) transport vesicles.

The p24 proteins seem to serve multiple roles, among them the structural organization of ER exit sites and the ER-Golgi intermediate compartment (25); the biogenesis of COPI and COPII vesicles (26, 27); and the selection of cargo proteins into these vesicles (especially glycosylphosphatidylinositol-anchored proteins (28). None of these roles is fully understood in their molecular mechanism. Additional postulated roles of TMED10 and other p24 proteins, for example in insulin secretion (29) and in micropinocytosis (30) (connected with vaccinia virus infection) may either be mediated directly by the p24 proteins themselves, or indirectly by other proteins that are localized through their function in the secretory pathway.

Since p24 proteins participate in many cellular functions, they are likely to play a role in disease in general, and in viral infection in particular. Our discovery of TMED10 as an essential ER anchor for gp40 marks the first time that a viral immunoevasin has been shown to use a p24 family member for its purposes. We believe that other MCMV and HCMV immunoevasins may do the same.

3.1.2.2 Interaction between gp40 and RAE-1g In the project proposed here, we will use our expertise to understand the molecular mechanism of action of gp40 upon its second target, RAE-1g.

25 TMED means 'Transmembrane emp24 domain-containing'. The TMED10 protein (UniProt P49755) is also called TMP21 or hp24d1. The p24 family is also called the Emp24 family. 26 Some exceptions to the ER/Golgi localization of the p24 proteins that were described in the literature might represent the physiological situation, or else they might be due to the saturation of the Golgi-to- ER retrieval mechanisms in the experimental systems.

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3.1.2.2.1 Cell surface downregulation of RAE-1g by gp40 in murine and human cells To replicate the data from the literature (20, 22), we co-transfected mouse fibroblast K41 cells with N-terminally HA-tagged RAE-1g (HA-RAE-1g) and gp40WT. As anticipated, we observed a loss of cell surface anti-HA staining compared to the control (HA-RAE-1g and empty vector) (Figure 3-4A). To study the interaction of gp40 with RAE-1g independent of the gp40-class I interaction, we decided to also use human cells, since gp40 does not bind to human class I molecules (18). We co-transfected human HEK293T (human embryonic kidney) cells with HA-RAE-1g and gp40WT, and we observed the same result as in the murine cells (Figure 3-4B). These results were confirmed with an anti-RAE-1g specific antibody, CX1 (Figure 3-4, bottom panels). Thus, we established the human HEK293T cells as a suitable model system27.

A B K41 (mouse) 293T (human) HA-RAE-1g HA-RAE-1g

293T HA K41 HA 1.01 1

0.80,8 0,8

0.60,6 0,6 HA

a 0.40,4 0,4

0.20,2 0,2

0.00 0 293T CX1 K41 CX1 1.01 1 0.80,8 0,8 0.60,6 0,6 CX1 Normalized cell surface level level surface cell Normalized 0.40,4 0,4

0.20,2 0,2

0.00 0 - gp40 + gp40 - gp40 + gp40

Figure 3-4. Preliminary data: gp40 downregulates cell surface levels of RAE-1g in murine and human cells. A) experiment performed in murine K41 cells. Mouse K41 fibroblasts were transduced with HA-RAE-1g alone (- gp40) or with gp40 wild type (+ gp40) and stained with an anti-HA antibody (aHA, top panel) or with the anti--RAE-1g antibody CX1 (bottom panel). The data confirm an efficient reduction of HA-RAE-1g surface expression by gp40 in murine cells. The normalized mean fluorescence intensity is shown (mean ± SEM, n = 3). B) experiment performed in human 293T cells. Human HEK293T cells were transfected with HA-RAE-1g alone (- gp40) or with gp40 wild type (+ gp40), and surface HA-RAE-1g was detected as in A. It shows that gp40 efficiently reduces HA-RAE-1g surface expression in human cells. (Mean ± SEM, n = 3)

27 gp40 functions in human cells since it binds to human TMED10 (our unpublished data).

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3.1.2.2.2 A second, independent, mechanism of RAE-1g epitope downregulation As described above [3.1.2.1.2], ER retention of the gp40/class I complex requires an additional host protein, TMED10. We therefore decided to test whether deletion of TMED10 prevents RAE-1g downregulation by gp40.

We used HEK293T TMED10-/- cells co-transfected with HA-RAE-1g and gp40WT, and we stained aliquots either with an anti-HA antibody to detect the N-terminal HA tag on RAE-1g, or with the RAE-1g-specific monoclonal antibody, CX1 (Figure 3-5A). Unexpectedly, the HA and CX1 stains were very different: the HA stain showed a cohort of RAE-1g that is present on the surface of TMED10-/- cells even in the presence of gp40WT (top row), but the surface RAE-1g was not as prominently recognised by CX1 (bottom row). We hypothesized that even though this portion of RAE-1g was not longer retained by gp40WT in the TMED10-/- cells, it was somehow masked from recognition by CX1, most likely by gp40 itself.

Since the difference between the HA and the CX1 stain in Figure 3-5A was only moderate in magnitude28, we decided to test the observation in an alternative system. We co-transfected HEK293T cells with HA-RAE-1g and either gp40WT or gp40LM (the linker mutant that does not bind to TMED10, [3.1.2.1.2]). Cell surface staining with anti-HA and CX1 antibodies showed a much stronger same effect: HA-RAE-1g is present on the cell surface, but it is not recognized by the conformation-specific antibody CX1 (Figure 3-5B). The simplest explanation of these data is that gp40LM travels to the cell surface in a complex with RAE-1g, and that at the cell surface, it masks RAE-1g such that the CX1 antibody cannot bind.

28 The reason for the moderate effect in the TMED10–/– cells may be twofold: first, we have observed that the TMED10–/– cells grow slowly (perhaps due to the important roles of the p24 proteins) and are very difficult to transfect efficiently. Second, other p24 family members are probably involved in the retention of gp40, and these other family members may have become overexpressed during the selection process in the generation of the TMED10–/– cells. See also the work program, 3.2.2.3.

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A B 293T TMED10-/- 293T WT HA-RAE-1g HA-RAE-1g KO HA 293T HA 293T HA 1.01 1 1

0.80,8 0,8 0,8

0.60,6 0,6 0,6 HA

a 0.40,4 0,4 0,4

0.20,2 0,2 0,2

0.00 0 0 KO CX1 293T CX1 293T CX1 1.01 1 1

0.80,8 0,8 0,8

0.60,6 0,6 0,6 CX1 Normalized cell surface level level surface cell Normalized 0.40,4 0,4 0,4

0.20,2 0,2 0,2

0.00 0 0 - gp40 + gp40WT - gp40 + gp40LM

Figure 3-5. Preliminary data: Cell surface gp40 masks RAE-1g from CX1 recognition. A) Experiment with gp40 wild type in TMED10-/- cells. HEK293T TMED10-/- cells were transfected with HA-RAE-1g alone (- gp40) or with gp40WT (+ gp40) and stained with an anti-HA antibody (aHA, top panel) or with the RAE-1g-specific CX1 antibody (bottom panel). It shows that the CX1 antibody recognizes surface HA-RAE-1g less efficiently than the anti-HA antibody. The mean fluorescence intensity is shown (mean ± SEM, n = 3). B) Experiment with the gp40 linker mutant in wild type cells. Wild type HEK293T cells were transfected with HA-RAE-1g alone (- gp40) or with the gp40 linker mutant (+ gp40LM) and stained for surface HA-RAE-1g as in A. The anti-HA stain confirms efficient surface expression of HA-RAE-1g while the CX1 stain does not, suggesting that gp40LM and CX1 compete for the same binding site in the RAE-1g molecule. (Mean ± SEM, n = 3)

3.1.2.3 Hypothesis: gp40 blocks the interaction between RAE-1g and NKG2D The idea that gp40 might bind to RAE-1g in such a way that it blocks its interaction with CX1 is supported by a crystal structure of the gp40/RAE-1g complex, where gp40 covers a large part of the extracellular domain of RAE-1g (Figure 3-6)29. Among the contact sites between gp40 and RAE-1g in the crystal structure is the P49LWY52 motif of RAE-1g (31). We assume that this PLWY motif is also part of the epitope of CX1 on RAE-1g30. This competition for binding to the PLWY motif explains why CX1 and gp40 cannot bind to RAE-1g at the same time.

Intriguingly, NKG2D binds to RAE-1b in the same way that gp40 binds to RAE-1g (32) (compared in Figure 3-6). This suggests that gp40 is incompatible not just with CX1

29 Importantly, binding of gp40 to RAE-1g has, so far, exclusively been shown with recombinant protein, but not in the cell. See our work program below [3.2.3.2]. 30 According to the manufacturer (Thermo Fisher Scientific), CX1 cross-reacts with RAE-1a and b, which have the PLWY motif, but not with RAE-1d and e, which do not have it. This suggests that the PLWY motif is part of the epitope.

96 3. Grant application binding but also with NKG2D binding. Thus, gp40 may bind to RAE-1g to sterically block and preclude NKG2D binding.

gp40 NKG2D

RAE-1g RAE-1b

Figure 3-6. gp40 might block the interaction between RAE-1g and NKG2D. Crystal structures of RAE-1g in complex with gp40 (left, (31)) and of RAE-1b with NKG2D (right, (32)). gp40 binds to RAE-1g in a similar fashion as does NKG2D to RAE-1b suggesting that gp40 binding might inhibit NKG2D binding to RAE-1g and thus block recognition of RAE-1g by NK cells.

To test this hypothesis, we performed a preliminary experiment: we stained wild type HEK293T cells expressing HA-RAE-1g and gp40LM with fluorescently labeled recombinant murine NKG2D and analyzed NKG2D binding by flow cytometry. The results were exactly as the staining data for the CX1 antibody in Figure 3-5B: even though HA-RAE-1g was present at the cell surface (as shown by the anti-HA stain), NKG2D could no longer bind to it (data not shown)31.

Based on these data and those described in 3.1.2.2.2, we therefore propose a novel additional mechanism of NK cell inhibition by gp40: we hypothesize that, in addition to retaining it inside the cell, gp40 blocks the interaction between RAE-1g and NKG2D by masking any RAE-1g molecules that are present on the cell surface (Figure 3-7).

31 The experiment is part of the work program (3.2.3.4.1) and described there in more detail.

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RAE-1g gp40 TMED10

1. 2. 3. 4. RAE-1g RAE-1g RAE-1g RAE-1g gp40WT gp40WT gp40LM TMED10-/-

Figure 3-7. Schematic summary of the two putative modes of action of gp40 on RAE-1g. 1. situation in the absence of gp40 expression. RAE-1g goes to the cell surface in the absence of gp40 and binds to NKG2D. 2. situation in the presence of gp40 and sufficient TMED10. gp40 tethers RAE-1g to TMED10 to retain it in the ER. 3. situation in TMED10-/- cells. In the absence of TMED10, gp40 binds to RAE-1g but can no longer retain it in the ER. 4. effect of the gp40 linker mutant (gp40LM). gp40LM binds to RAE-1g but is unable to bind to TMED10 and can thus no longer retain RAE-1g in the ER. In cases 3 and 4, a complex of RAE-1g and gp40 reaches the cell surface, but NKG2D cannot bind to RAE-1g since the binding site is covered by gp40.

During infection, the majority of RAE-1g is probably retained inside the cells, but a cohort of RAE-1g might escape gp40-mediated retention either because it already existed on the cell surface by the time that gp40 was synthesized, or because there is not enough TMED10 present in the cell to anchor all the gp40 molecules in the ER. In our preliminary experiments, we have indeed seen gp40WT, expressed from a viral promoter, at the surface of transduced cells (data not shown)32. During CMV infection the secretory pathway is remodelled to yield a new viral assembly compartment made from cellular organelles including ER, Golgi network and endosomes, and the cellular localization of host proteins, including p24 proteins, is influenced by the infection (33, 34, 35); this may further increase the chances of gp40 traveling to the cell surface.

32 Part of the work program, see 3.2.3.5.1.

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3.1.3 Project-related publications

3.1.3.1 Articles published by outlets with scientific quality assurance, book publications, and works accepted for publication but not yet published • V. R. Ramnarayan, L. Janßen, Z. Hein, N. Lis, S. Ghanwat, and S. Springer: Cytomegalovirus gp40/m152 uses TMED10 as ER anchor to retain MHC class I. Cell Reports 23 (2018), 3068-3077; doi: 10.1016/j.celrep.2018.05.017

• L. Janssen, V. Ramnarayan, M. Aboelmagd, M. Iliopoulou, Z. Hein, I. Majoul, S. Fritzsche, A. Halenius, and S. Springer: The murine cytomegalovirus immunoevasin gp40 binds MHC class I molecules to retain them in the early secretory pathway. J. Cell Sci. 129 (2016), 219-227; doi: 10.1242/jcs.175620.

• S. Fritzsche, E. Tolba Abualrous, B. Borchert, F. Momburg, and S. Springer: Allotype-specific release from endoplasmic reticulum matrix proteins controls cell surface transport of MHC class I molecules. Traffic 16 (2015), p. 591-603; doi: 10.1111/tra.12279

• S. Fritzsche and S. Springer: Investigating MHC class I folding and trafficking with pulse-chase experiments. Mol. Immunol. 55 (2013), p. 126-130.

• C. Howe*, M. Garstka*, M. Al-Balushi, E. Ghanem, A. N. Antoniou, S. Fritzsche, G. Jankevicius, N. Kontouli, C. Schneeweiss, A. Williams, T. Elliott, and S. Springer: Calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules. EMBO Journal 28 (2009), p. 3730-3744.

• M. Garstka*, M. Borchert*, M. Al-Balushi*, P.V.K. Praveen, N. M. Kühl, I. Majoul, R. Duden, and S. Springer: Peptide-receptive MHC class I molecules cycle between endoplasmic reticulum and cis-Golgi in wild type lymphocytes. J. Biol. Chem. 282 (2007), p. 30680-90.

3.1.3.2 Other publications – None.

3.1.3.3 Patents – None.

3.2 Objectives and work program We will explain the molecular mechanism of gp40-mediated RAE-1g downregulation in the presence and absence of sufficient TMED10 with individual proteins expressed in cells, and in an infection model. If time allows, we will also apply our technical

99 3. Grant application expertise to investigate the downregulation of the human NKG2D ligands ULBP1, ULBP2, and MICB by the HCMV immunoevasin UL16.

3.2.1 Anticipated total duration of the project This application builds on our previous project, SP 583/11-1 "Molecular cell biology of MHC class I retention by MCMV gp40", and three more years.

3.2.2 Objectives We will ask the following specific questions, which are directly mirrored in the work program below [3.2.3]:

3.2.2.1 Influence of gp40 on maturation and trafficking of RAE-1g • What is the intracellular trafficking pattern and dynamic localization of RAE-1g? • Does gp40 retain RAE-1g in the ER, with eventual degradation by the proteasome, or does it direct it towards lysosomal degradation?

3.2.2.2 Protein complexes during RAE-1g downregulation • Do RAE-1g and gp40 bind to each other in the cell? • Do RAE-1g and gp40 traffic together in the secretory pathway? • Does gp40 bind RAE-1g and MHC class I simultaneously or separately?

3.2.2.3 Role of TMED10 and other p24 proteins in RAE-1g downregulation • Are TMED10 and other p24 proteins required for RAE-1g downregulation by gp40? • Do TMED10 and/or other p24 proteins influence trafficking of RAE-1g in the presence of gp40? • Does TMED10 or another p24 protein form a complex with gp40, or RAE-1g or both?

3.2.2.4 gp40-mediated masking of cell surface RAE-1g and inhibition of NKG2D binding • Does gp40 prevent binding between RAE-1g and NKG2D on the cell surface, as hypothesized in 3.1.2.3? • Does this effect influence NK cell activation?

3.2.2.5 gp40 and RAE-1g interaction during MCMV infection • How strong is RAE-1g downregulation during MCMV infection?

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• Are the retention factors (TMED10 or other p24 protein) out-titrated by gp40 during MCMV infection? • Is the cell surface masking effect used by gp40 during infection? • Is the trafficking of RAE-1g and gp40 the same during MCMV infection as when proteins are expressed in uninfected cells?

3.2.2.6 Human NKG2D ligands and HCMV immunoevasins The human cytomegalovirus (HCMV) also codes for immunoevasins that block the function of NKG2D ligands. For the immunoevasin UL16, it was previously shown that it retains ULBP1, ULBP2, and MICB in the cell interior. However, it is unknown how UL16 itself is retained in the cell (since, just as gp40, it does not have any known retention motif) and it is also unknown whether a fraction of the UL16/NKG2D ligand complexes can reach the cell surface during infection (12, 13, 36).

We hypothesize that HCMV UL16 might work in a way similar to MCMV gp40, and thus we will apply the same approach to elucidate its mode of action33. This work will be carried out if time and resources allow.

We will aim to answer following questions:

• Does UL16 bind to the NKG2D ligands (ULBP1, ULBP2, and MICB) that it retains inside the cell? • Does UL16 bind to p24 family members? • If yes, then is this binding required for UL16 to retain the NKG2D ligands? • Does UL16 mask one or more of these NKG2D ligands at the cell surface according to our gp40/RAE-1g hypothesis [3.1.2.3]?

3.2.3 Work program including proposed research methods The individual subchapters of this section correspond to the subchapters of the Aims section (for example, 3.2.3.1 is the work package that corresponds to aim 3.2.2.1). Figure 3-8 shows an overview of the work program and the connections between the work packages.

33 UL142 is another HCMV immunoevasin that downregulates NKG2D ligands (see 3.1.1.2.2). It is conceivable to carry out a similar investigation as described here on UL142.

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Figure 3-8. Work program. The text written in black font show the work packages, in red the conclusions deduced from their outcomes (solid arrows). By comparing the mode of action of gp40 with that one of UL16, we will eventually describe analogies between murine and human CMV immunoevasion strategies (dotted arrows).

3.2.3.1 Work program for 3.2.2.1: Influence of gp40 on maturation and trafficking of RAE-1g We hypothesize that gp40WT retains HA-RAE-1g in the early secretory pathway, i.e. in the ER or the cis-Golgi, or by circulating between these two compartments. Therefore, we will investigate the dynamics of maturation, intracellular transport, and degradation of RAE-1g using radioactive labeling and pulse-chase experiments combined with glycan digestion. We will confirm the steady-state intracellular localization using immunofluorescence microscopy34.

As a next step, we will test how these parameters are influenced by the presence of gp40, and especially whether HA-RAE-1g is now retained in the early secretory pathway.

34 Both methods, radioactive pulse-chase and immunofluorescence microscopy, are well established in our group (17, 18, 37).

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If our studies do not confirm the retention of HA-RAE-1g by gp40WT, we will investigate whether HA-RAE-1g downregulation is a result of protein degradation. We will use lysosomal inhibitors (leupeptin or chloroquine) or proteasomal inhibitors (MG132 or lactacystin) and check by flow cytometry whether HA-RAE-1g cell surface levels are rescued.

3.2.3.2 Work program for 3.2.2.2: Protein complexes during RAE-1g downregulation RAE-1g and gp40 were shown to co-crystallize (31), but they have not been shown to interact in cells. We hypothesize that HA-RAE-1g and gp40WT form a complex within the cell and that both proteins are retained in the early secretory pathway.

Therefore, we will test whether gp40WT and HA-RAE-1g, expressed in HEK293T cells, can be co-immunoprecipitated after cell lysis with digitonin, a mild detergent that does not disrupt protein-protein interactions.

Another set of co-immunoprecipitations will be performed in mouse cells transfected with gp40WT or with HA-RAE-1g or both. This will inform us if gp40 can bind to both HA-RAE-1g and class I at the same time, or to only one of these targets at a time. Using different lysis conditions (such as the more disruptive detergent Triton X-100) we will estimate whether gp40 binds to class I and to HA-RAE-1g with the same, or with different, affinities35,36.

We will also perform radioactive pulse-chase and re-immunoprecipitation experiments37 to find out whether gp40WT and HA-RAE-1g bind already in the ER or only in the Golgi, and whether they traffic in the cell together38.

If the experiments in 3.2.3.1 show that gp40 does not, or not fully, retain HA-RAE-1g but triggers its lysosomal or proteosomal degradation, then we will use

35 gp40 and murine MHC class I molecules co-immunopecipitate in digitonin. It has never been tested whether this interaction persists in Triton X-100 (17). 36 Our result will only show the preference for binding. The exact binding affinity should be measured with recombinant proteins by surface plasmon resonance or isothermal titration calorimetry (31). This is not planned in this project. 37 This re-immunoprecipitation experiment in detail: If class I, gp40, and RAE-1g form a trimeric complex, then in the presence of gp40 (but not in its absence), RAE-1g should co-precipitate with class I. Thus, after digitonin lysis, class I will be immunoprecipitated (for example H-2Db with the monoclonal antibody 28-14-8S), the immunoprecipitates will be boiled in sodium dodecyl sulfate (SDS) buffer to dissolve them, and HA-RAE-1g will be re-precipitated from the supernatant. 38 We have shown, in an analogous experiment, early binding of gp40 to class I and exit of the complex from the ER to the cis-Golgi in (17).

103 3. Grant application immunofluorence microscopy to confirm that during lysosomal or proteosomal inhibition, gp40 and HA-RAE-1g co-localize with lysosomal or proteosomal markers, respectively.

3.2.3.3 Work program for 3.2.2.3: Role of TMED10 and other p24 proteins in RAE-1g downregulation We have found in preliminary experiments that knockdown or deletion of TMED10 partially rescues RAE-1g surface expression [3.1.2.1.2, Figures 3-3 and 3-5], and we hypothesize that TMED10 binds to gp40 and causes its retention, which then causes retention of RAE-1g. The p24 family consists of ten highly homologous members that stabilize each other by forming heterodimeric and heterotetrameric complexes [3.1.2.1.3]. Thus, the knockdown of one gene may have direct but also indirect effects through the loss of another p24 family member, and in addition to TMED10, other p24 proteins may be involved in the retention of gp4039.

3.2.3.3.1 Influence of p24 proteins on gp40-mediated RAE-1g retention We will confirm the TMED10 data and extend them to other members of the p24 family, initially with small interfering RNA (siRNA)40 against p24 gene expression in cells that express HA-RAE-1g and gp40WT. With flow cytometry, we will test whether such knockdowns rescue the cell surface levels of HA-RAE-1g. If this experiment points to another p24 protein besides TMED10 with a role in RAE-1g downregulation, then we will test it with the same set of experiments described in the following for TMED10, including gene knockdowns.

3.2.3.3.2 Influence of p24 proteins on RAE-1g trafficking dynamics and steady-state localization To estimate the influence of TMED10 on the dynamics of trafficking and retention of RAE-1g, we will perform radioactive pulse-chase experiments of HA-RAE-1g alone or in the presence of gp40WT as described in 3.2.3.1, but now additionally in TMED10-/- cells (Figure 3-7). We will complement the pulse-chase results with

39 This important question has not been conclusively addressed in our previous work on MHC class I retention of gp40 [3.1.2.1.1](18). 40 We believe that for the p24 family, gene knockdowns are still preferable to knockouts, since during the selection processes for gene knockouts, cell line variants may be positively selected – due to the essential nature of the p24 genes – that compensate for the loss of one p24 protein by spontaneous overexpression of one or more of the other family members, obscuring the phenotype.

104 3. Grant application immunofluorescence microscopy, using organelle markers to test whether TMED10, gp40WT, and HA-RAE-1g co-localize in the same cellular compartment.

3.2.3.3.3 Specific use of the gp40 linker mutant p24 proteins play a central role in COPI- and COPII-mediated ER-Golgi-ER transport processes, especially for GPI-anchored proteins (24). Thus, knockdowns of one or more p24 genes, such as TMED10, might negatively affect cell viability or disturb specific aspects of protein trafficking, making such mutant cell lines unsuitable for the study of retention phenomena, while knockouts might fail to show the true phenotype40. Since gp40LM no longer binds to TMED10 [3.1.2.1.2], we think that using gp40LM in wild type cells might give more reliable results than using gp40WT in TMED10-/- cells for the study of the influence of TMED10 binding on the dynamics of trafficking and retention of RAE-1g.

We will therefore perform pulse-chase and microscopy experiments with gp40LM analogous to those in 3.2.3.3.2, and compare carefully.

3.2.3.3.4 Binding of gp40 to TMED10 and RAE-1g To test whether gp40 binds to RAE-1g in cells, we will perform co-immunoprecipitations. To test whether TMED10, gp40, and RAE-1g form a trimeric complex, we will perform a re-immunoprecipitation experiment41.

3.2.3.4 Work program for 3.2.2.4: gp40-mediated masking of cell surface RAE-1g and inhibition of NKG2D binding In preliminary experiments, we have found that in the presence of gp40, cell surface HA-RAE-1g is no longer detected by the CX1 antibody. We hypothesize that cell surface gp40 masks cell surface HA-RAE-1g by covering the CX1 epitope, which may correspond to the interaction surface with NKG2D (Figure 3-6), to inhibit NKG2D binding [3.1.2.3]. Thus, for gp40LM in wild type cells (or for gp40WT in

41 Re-immunoprecipitation experiment in detail: If TMED10, gp40, and RAE-g form a trimeric complex, then in the presence of gp40 (but not in its absence), HA-RAE-1g should co-precipitate with TMED10. Thus, after digitonin lysis, HA-RAE-1g will be immunoprecipitated with an anti-HA monoclonal antibody, the immunoprecipitates will be boiled in SDS buffer to dissolve them, and TMED10 will be re-precipitated from the supernatant. We have ample experience with re-immunoprecipitation experiments (17); an analogous experiment can also be done by immunoprecipitation and subsequent Western blotting.

105 3. Grant application

TMED10-/- cells), we expect to see reduced NKG2D binding and NK cell activation, even though HA-RAE-1g is present on the cell surface.

3.2.3.4.1 Testing NKG2D binding to the gp40/RAE-1g complex For these experiments, we will again generate conditions in which gp40 is not anchored in the ER by TMED10. This will be achieved in two different experimental systems: with gp40WT in TMED10-/- cells, and with gp40LM in wild type cells (which may be better, see 3.2.3.3.3).

We will express HA-RAE-1g in these cells and stain the cell surface with anti-HA antibody and CX1 (as in the preliminary experiments, 3.1.2.2.2), and also with a recombinant mouse NKG2D/IgFc chimera protein42. If our hypothesis is correct, then we will see anti-HA staining, indicating the presence of HA-RAE-1g at the cell surface, but no NKG2D staining, indicating the masking of the extracellular part of HA-RAE-1g by gp40, and the blocking of its interaction with CX1 and with the NKG2D receptor.

3.2.3.4.2 Testing NK cell activation by the gp40/RAE-1g complex Our group has no prior practical experience with primary NK cells. Thus, to test NK cell activation, we will enter into a collaboration with Dr. Benedict Chambers at the Karolinska Institute in Stockholm. Dr. Chambers has wide expertise in NK cell cultivation and activation assays, and the department is famous for exploring NK cell biology43. To measure NK cell activation, we will use assays for CD107a degranulation and interferon-g release (to monitor NK cell activation) and/or chromium release assays to monitor target cell killing44. The experiments will be done

42 This fusion protein of the extracellular domain of murine NKG2D and the Fc domain of an antibody is available from R&D systems (cat. nr. 139-NK-050). It can be used like a primary antibody in flow cytometry staining to test for the interaction of NKG2D with RAE-1. If the staining is too weak, the protein can be biotinylated and tetramerized with streptavidin to make use of the avidity effect (38) , as with MHC class I tetramers. 43 A description of Benedict Chambers' work and relevant publications is found at https://ki.se/en/medh/benedict-chambers-group; a letter of collaboration is attached. 44 CD107a degranulation assay: CD107a, also known as LAMP-1 (lysosomal- associated membrane protein 1), is a marker for NK cell degranulation that is upregulated in activated NK cells. Expression of CD107a is correlated with NK cell cytotoxicity and can be quantified by cell surface staining and flow cytometry analysis [39]. For each experiment, we will measure specific and spontaneous degranulation. IFN-γ release assay: upon activation, NK cells release the cytokine interferon- gamma (IFN-γ) that is critical for the innate and adaptive immune responses ([40]). We will measure IFNγ release by NK cells using a sandwich ELISA.

106 3. Grant application in the murine cell line RMA-S, which is widely used as a target to study NK cell activation.

3.2.3.4.3 Measuring cell surface levels of gp40WT Our hypothesis of the masking of surface RAE-1g by gp40 as a second, independent mechanism of action of gp40 on RAE-1g [3.1.2.2.2] implies that during infection, in wild type cells, wild type gp40 can (at least in some cases) escape TMED10-mediated retention to reach the cell surface and act on pre-existing or escaped RAE-1g [see 3.1.2.3].

To test whether this is the case in cell culture, we will use an antibody that recognizes the extracellular domain of gp4045, such that it can be detected by flow cytometry. We will analyze the cell surface levels of gp40WT and gp40LM in wild type and in TMED10-/- cells in the presence and absence of RAE-1g.

3.2.3.5 Work program for 3.3.2.2.5: gp40 and RAE-1g interaction during MCMV infection We also hypothesize that during MCMV infection, some gp40 molecules and/or gp40/RAE-1g complexes escape the ER and reach the cell surface. This is possible if not enough TMED10 is available [3.1.2.3]. The cohort of RAE-1g that is present on the surface of such infected cells would then still not be able to bind to NKG2D, since it is masked by gp40.

3.2.3.5.1 Detection of HA-RAE-1g/gp40 complexes at the surface of infected cells To test whether gp40 is visible at the cell surface during MCMV infection, we will infect K41/HA-RAE-1gcells (mouse fibroblasts that express endogenous class I and transduced HA-RAE-1g) and B78H1/HA-RAE-1gcells (mouse melanoma cells without endogenous class I and transduced HA-RAE-1g) with wild type MCMV that expresses GFP as an infection marker (GFP-MCMV-WT in the following). We will stain for surface gp40 as above [3.2.3.4.3]46.

Chromium release assay: this assay will allow us to reaffirm cytotoxicity of NK cells toward target cells. After labeling with 51Cr, target cells are mixed with NK cells. As a result of NK cell activation, target cells are lysed, and 51Cr is released into the supernatant [41]. 45 The m152.05 antibody from the Center for Proteomics, Rijeka, Croatia (Catalog No.: HR-MCMV-11). 46 We will also stain with HA and CX1 to test for RAE-1g surface expression and CX1 accessibility; and with the murine anti-class I antibodies B22.249 (H-2Db) and Y3 (H-2Kb). An additional control will be the m152-deleted virus GFP-MCMV-∆m152.

107 3. Grant application

The GFP-MCMV-WT infections will be done at different multiplicities of infection (MOI). We hypothesize that at higher MOIs, TMED10 will be out-titrated by gp40 (i.e., the amount of gp40 in the cell will exceed the available TMED10), and increasing amounts of gp40, gp40/HA-RAE-1gcomplexes, and/or gp40/MHC class I complexes will escape retention47.

As a control for TMED10 out-titration, we will use TMED10-/- cells [3.2.3.3.2] for infection. To show whether the surface expression of gp40 has an effect on NKG2D recognition of surface HA-RAE-1g, we will stain with recombinant NKG2D as in 3.2.3.4.1.

3.2.3.5.2 Trafficking of RAE-1g during MCMV infection We will investigate trafficking of HA-RAE-1g in MCMV-infected cells by pulse-chase and immunofluorescence microscopy in analogy to 3.2.3.1.

3.2.3.6 Work program for 3.2.2.6: Human NKG2D ligands and HCMV immunoevasins If time and resources of the project are sufficient, we will also investigate the molecular mechanism of action of UL16 on human NKG2D ligands, since it might be similar to the mechanism of action of gp40 on the murine NKG2D ligands.

We will establish human cell lines that express UL16 and N-terminally HA-tagged MICB, ULBP1, or ULBP2. We will test intracellular retention of the NKG2D ligands by flow cytometry.

To find whether UL16 binds to the NKG2D ligands and/or to p24 proteins, we will perform digitonin cell lysis and co-immunoprecipitation (analogous to 3.2.2.2 and 3.2.3.3.4).

To find whether p24 proteins are necessary for intracellular retention of the NKG2D ligands by UL16, we will perform siRNA knockdowns and anti-HA flow cytometry (analogous to 3.2.3.3.2). If we find that this is the case, then it will be very interesting to delineate the p24-binding region of UL16 (in analogy to the useful linker mutant of gp40, 3.2.3.3.3), if time allows.

47 Should this not be the case, then we will test whether cellular TMED10 and other p24 protein levels are upregulated during virus infection. This would be remarkable, since p24 are considered household genes. We will test by quantitative real-time PCR and by Western blotting.

108 3. Grant application

Finally, we will test, using staining with recombinant NKG2D48 staining, whether NKG2D binding to extracellular domains of ULBP1, ULBP2 and MICB is blocked by UL16 on the cell surface by comparing their surface staining with that of an anti-HA antibody (in analogy to 3.2.3.4.1).

3.2.4 Data handling Handling and storage of data will be according to the institutional guidelines of Jacobs University Bremen. Primary data will be kept on record for 15 years and made available to any interested party upon request.

3.2.5 Other information: Not applicable.

3.2.6 Description of proposed investigations involving experiments on humans, human materials, or animals as well as dual use research of concern: Not applicable.

3.2.7 Information on scientific and financial involvement of international cooperation partners: Not applicable.

3.3 Bibliography 1 M. J. Smyth et al., “Activation of NK cell cytotoxicity,” in Molecular Immunology, 2005, vol. 42, no. 4 SPEC. ISS., pp. 501–510. 2 K. D. Cook, S. N. Waggoner, and J. K. Whitmire, “NK cells and their ability to modulate T cells during virus infections.,” Crit. Rev. Immunol., vol. 34, no. 5, pp. 359–88, 2014. 3 A. Cerwenka and L. L. Lanier, “Natural killer cells, viruses and cancer,” Nat. Rev. Immunol., vol. 1, no. 1, pp. 41–49, 2001. 4 D. Schmiedel and O. Mandelboim, “NKG2D ligands-critical targets for cancer immune escape and therapy,” Front. Immunol., vol. 9, no. SEP, pp. 1–10, 2018. 5 A. Zingoni et al., “NKG2D and its ligands: ‘One for all, all for one,’” Front. Immunol., vol. 9, no. MAR, 2018. 6 D. H. Raulet, “Roles of the NKG2D immunoreceptor and its ligands,” Nat. Rev. Immunol., vol. 3, no. 10, pp. 781–790, 2003. 7 M. Hummel and M. M. Abecassis, “A model for reactivation of CMV from latency.,” J. Clin. Virol., vol. 25 Suppl 2, pp. S123-36, 2002. 8 I. Brizić, B. Lisnić, W. Brune, H. Hengel, and S. Jonjić, “Cytomegalovirus

48 This fusion protein of the extracellular domain of human NKG2D and the Fc domain of an antibody is available from R&D systems (cat. nr. 1299-NK-050).

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Infection: Mouse Model,” Curr. Protoc. Immunol., vol. 122, no. 1, p. e51, 2018. 9 A. Halenius, C. Gerke, and H. Hengel, “Classical and non-classical MHC i molecule manipulation by human cytomegalovirus: So many targets - But how many arrows in the quiver?,” Cell. Mol. Immunol., vol. 12, no. 2, pp. 139–153, 2015. 10 M. Reddehase and N. Lemmermann, “Mouse Model of Cytomegalovirus Disease and Immunotherapy in the Immunocompromised Host: Predictions for Medical Translation that Survived the ‘Test of Time,’” Viruses, vol. 10, no. 12, p. 693, 2018. 11 O. Ashiru, N. J. Bennett, L. H. Boyle, M. Thomas, J. Trowsdale, and M. R. Wills, “NKG2D ligand MICA is retained in the cis- by human cytomegalovirus protein UL142.,” J. Virol., vol. 83, no. 23, pp. 12345–12354, 2009. 12 C. Dunn et al., “Human Cytomegalovirus Glycoprotein UL16 Causes Intracellular Sequestration of NKG2D Ligands, Protecting Against Natural Killer Cell Cytotoxicity,” J. Exp. Med., vol. 197, no. 11, pp. 1427–1439, 2003. 13 A. Rolle et al., “Effects of Human Cytomegalovirus Infection on Ligands for the Activating NKG2D Receptor of NK cells: Up-Regulation of UL16-Binding Protein (ULBP)1 and ULBP2 Is Counteracted by the Viral UL16 Protein,” J. Immunol., vol. 171, no. 2, pp. 902–908, 2003. 14 O. Ashiru, N. J. Bennett, L. H. Boyle, M. Thomas, J. Trowsdale, and M. R. Wills, “NKG2D ligand MICA is retained in the cis-Golgi apparatus by human cytomegalovirus protein UL142,” J Virol, vol. 83, no. 23, pp. 12345–12354, 2009. 15 M. Hasan et al., “Selective Down-Regulation of the NKG2D Ligand H60 by Mouse Cytomegalovirus m155 Glycoprotein,” J. Virol., vol. 79, no. 5, p. 2920 LP-2930, Mar. 2005. 16 A. Krmpotic et al., “ARTICLE NK cell activation through the NKG2D ligand MULT-1 is selectively prevented by the glycoprotein encoded by mouse cytomegalovirus gene m145,” vol. 201, no. 2, pp. 211–220, 2005. 17 L. Janßen et al., “The murine cytomegalovirus immunoevasin gp40 binds MHC class I molecules to retain them in the early secretory pathway,” J. Cell Sci., vol. 129, no. 1, pp. 219–227, 2016. 18 V. R. Ramnarayan, Z. Hein, L. Janßen, N. Lis, S. Ghanwat, and S. Springer, “Cytomegalovirus gp40/m152 Uses TMED10 as ER Anchor to Retain MHC Class I,” Cell Rep., vol. 23, no. 10, pp. 3068–3077, 2018. 19 A. Krmpotić et al., “MCMV glycoprotein gp40 confers virus resistance to CD8+ T cells and NK cells in vivo,” Nat. Immunol., vol. 3, no. 6, pp. 529–535, 2002. 20 M. Lodoen et al., “NKG2D-mediated Natural Killer Cell Protection Against Cytomegalovirus Is Impaired by Viral gp40 Modulation of Retinoic Acid Early Inducible 1 Gene Molecules,” J. Exp. Med., vol. 197, no. 10, pp. 1245–1253, 2003. 21 L. Zhi et al., “Direct interaction of the mouse cytomegalovirus m152/gp40 immunoevasin with RAE-1 isoforms,” Biochemistry, vol. 49, no. 11, pp. 2443– 2453, 2010.

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22 J. Arapovic et al., “Differential Susceptibility of RAE-1 Isoforms to Mouse Cytomegalovirus,” J. Virol., vol. 83, no. 16, pp. 8198–8207, 2009. 23 H. Ziegler, W. Muranyi, H. G. Burgert, E. Kremmer, and U. H. Koszinowski, “The luminal part of the murine cytomegalovirus glycoprotein gp40 catalyzes the retention of MHC class I molecules,” EMBO J., vol. 19, no. 5, pp. 870–881, 2000. 24 N. Pastor-Cantizano, J. C. Montesinos, C. Bernat-Silvestre, M. J. Marcote, and F. Aniento, “P24 Family Proteins: Key Players in the Regulation of Trafficking Along the Secretory Pathway,” Protoplasma, vol. 253, no. 4, pp. 967–985, 2016. 25 S. Mitrovic, H. Ben-Tekaya, E. Koegler, J. Gruenberg, and H.-P. Hauri, “The Cargo Receptors Surf4, Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC)-53, and p25 Are Required to Maintain the Architecture of ERGIC and Golgi,” Mol. Biol. Cell, vol. 19, pp. 1976–1990, 2008. 26 J. G. D’Arcangelo et al., “Traffic of p24 proteins and COPII coat composition mutually influence membrane scaffolding,” Curr. Biol., vol. 25, no. 10, pp. 1296– 1305, 2015. 27 L. P. Jackson, “Structure and mechanism of COPI vesicle biogenesis,” Curr. Opin. Cell Biol., vol. 29, no. 1, pp. 67–73, 2014. 28 A. Copic, C. F. Latham, M. A. Horlbeck, J. G. D’Arcangelo, and E. A. Miller, “ER Cargo Properties Specify a Requirement for COPII Coat Rigidity Mediated by Sec13p,” Science (80-. )., vol. 335, no. March, pp. 1359–1363, 2012. 29 L. Zhang and A. Volchuk, “P24 family type 1 transmembrane proteins are required for insulin biosynthesis and secretion in pancreatic β-cells,” FEBS Lett., vol. 584, no. 11, pp. 2298–2304, 2010. 30 R. D. Luteijn et al., “A genome-wide haploid genetic screen for essential factors in vaccinia virus infection identifies TMED10 as regulator of macropinocytosis,” 2018. 31 R. Wang et al., “Structural basis of mouse cytomegalovirus m152/gp40 interaction with RAE1 reveals a paradigm for MHC/MHC interaction in immune evasion,” Proc. Natl. Acad. Sci., vol. 109, no. 51, pp. E3578–E3587, 2012.

32 P. Li, G. Mcdermott, and R. K. Strong, “Crystal Structures of RAE-1 ␤ and Its Complex with the Activating Immunoreceptor NKG2D,” vol. 16, pp. 77–86, 2002. 33 R. Tandon and E. S. Mocarski, “Viral and host control of cytomegalovirus maturation,” Trends Microbiol., vol. 20, no. 8, pp. 392–401, 2012. 34 K. Nightingale et al., “High-Definition Analysis of Host Protein Stability during Human Cytomegalovirus Infection Reveals Antiviral Factors and Viral Evasion Mechanisms,” Cell Host Microbe, vol. 24, no. 3, p. 447–460.e11, 2018. 35 P. Lučin et al., “Cytomegaloviruses Exploit Recycling Rab Proteins in the Sequential Establishment of the Assembly Compartment,” Front. Cell Dev. Biol., vol. 6, no. December, pp. 4–11, 2018. 36 S. A. Welte et al., “Selective intracellular retention of virally induced NKG2D ligands by the human cytomegalovirus UL16 glycoprotein,” Eur. J. Immunol.,

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vol. 33, no. 1, pp. 194–203, 2003. 37 S. Fritzsche and S. Springer, “Pulse-chase analysis for studying protein synthesis and maturation,” Curr. Protoc. Protein Sci., vol. 2014, no. November, p. 30.3.1-30.3.23, 2014. 38 A. Diefenbach, A. M. Jamieson, S. D. Liu, N. Shastri, and D. H. Raulet, “Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages,” Nat. Immunol., vol. 1, p. 119, Aug. 2000. 39 G. Alter, J. M. Malenfant, and M. Altfeld, “CD107a as a functional marker for the identification of natural killer cell activity,” J. Immunol. Methods, vol. 294, no. 1–2, pp. 15–22, 2004. 40 A. Lalvani and M. Pareek, “Interferon gamma release assays: principles and practice,” Enferm. Infecc. Microbiol. Clin., vol. 28, no. 4, pp. 245–252, 2010. 41 M. A. Karimi et al., “Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay,” PLoS One, vol. 9, no. 2, 2014.

3.4 Requested modules/funds

3.4.1 Basic Module

3.4.2 Funding for staff For realization of the project within the requested duration of the funding, one postdoctoral associate position (100% TVöD E13; approx. € 72 000 p.a.49) is necessary. This position will be filled by Natalia Lis, who has worked on gp40 and MHC class I during her PhD, and who has generated the preliminary data for this application. She is familiar with the methods used, and she can start the project immediately. Her CV and publication record are attached.

Since Jacobs University professors are not provided with academic research staff, a postdoctoral position is especially important for the success of the project. A PhD student would not be able to perform the project successfully.

Additionally, we request one student assistant position for three years (13 Euros per hour × 35 hours per month × 12 months= 5460 Euros p.a.) who will support Ms Lis by carrying out technical routine work such as simple cloning experiments or cell culture maintenance.

49 Salaries are from the document "Personalmittelsätze der DFG für das Jahr 2018”; numbers have been rounded.

112 3. Grant application

The work schedule is shown in Table 3-1. Handling of the work package 3.2.3.6 depends on the time and resources left and is thus depicted in grey.

Table 3-1. Work schedule of the project

Quarter ð 1 2 3 4 5 6 7 8 9 10 11 12 Work package ò

3.2.3.1. RAE-1g downregulation 3.2.3.2. Protein complexes 3.2.3.3. Role of p24 proteins 3.2.3.4. Masking of surface RAE-1g 3.2.3.5. MCMV infection 3.2.3.6. Human NKG2D ligands

3.4.2.1 Project costs 3.4.2.1.1 Equipment up to Euro 10,000, Software and Consumables We request a total of 75 000 Euros for consumables (25 000 Euros p.a.) detailed as below (Table 3-2).

Table 3-2. Costs for consumables (Euros p.a.)

Items Costs € p.a.

Molecular biology consumables (e.g. plasmids, restriction enzymes, siRNA kits, 6 000 sequencing analysis, bacterial media) Cell culture consumables (e.g. plastics, media, reagents) 9 000 Flow cytometry consumables (antibodies and reagents) 4 000 Microscopy consumables (antibodies and reagents) 3 000 Gene synthesis 2 000 Radioactive cysteine/methionine 1 000 Sum p.a. 25 000

3.4.2.1.2 Travel expenses For travel to conferences50, we anticipate 1 500 Euros p.a.

50 We will probably attend the following meetings: Mosbacher Kolloquium of the Gesellschaft für Biochemie und Molekularbiologie, the meetings of the Deutsche Gesellschaft für Zellbiologie, and 1-2 meetings of the German Society for Immunology.

113 3. Grant application

3.4.2.1.3 Project-related publication expenses We anticipate 750 Euros for each year of the project, together 2 250 Euros.

3.4.3 Total costs For each year, the total costs (in Euro) are summarized in Table 3-3.

Table 3-3. Total costs of the project

Position Keyword Year 1 Year 2 Year 3 Sum

3.4.1 Staff 77 460 77 460 77 460 232 380 3.4.2 Consumables 25 000 25 000 25 000 75 000 3.4.3 Travel 1 500 1 500 1 500 4 500 3.4.4 Publication 750 750 750 2 250 3.4.5 Others - - - - Sum 104 710 104 710 104 710 314 130 Overhead 22% 23 036 23 036 23 036 69 109 Total costs 127 746 127 746 127 746 383 238

3.5 Project requirements

3.5.1 Employment status information Sebastian Springer is Full Professor at Jacobs University Bremen. He has an unlimited contract.

3.5.2 First-time proposal data: Not applicable.

3.5.3 Composition of the project group The project group will consist of the postdoctoral fellow, Dr. Natalia Lis. Prof. Springer will dedicate 20% of his time to the project.

3.5.4 Cooperation with other researchers

3.5.4.1 Researchers with whom we have agreed to cooperate on the project Benedict Chambers, Karolinska Institute, Stockholm.

114 3. Grant application

3.5.4.2 Researchers with whom we have collaborated scientifically within the past three years Adnane Achour, Stockholm; Tim Elliott, Southampton; Sine Hadrup, Copenhagen; Hartmut Hengel and Anne Halenius, Freiburg; Robert Tampé, Frankfurt; Martin Messerle, Hannover; Melanie Brinkmann, Braunschweig.

3.5.5 Scientific equipment The following equipment that is essential for the project is available and maintained by the university: Confocal laser scanning microscopy; Flow cytometry.

3.5.6 Project-relevant cooperation with commercial enterprises: None.

3.5.7 Project-relevant participation in commercial enterprises: None.

3.5.8 Additional information All permits for the genetic engineering work are in place at Jacobs University (project leader: Sebastian Springer). This project is not the subject of any other application for funding.

115 4. Manuscript

4 Manuscript

The following manuscript contains the majority of the results obtained during my PhD. It was submitted to PLOS Pathogens in July, 2020. In November 2020 the manuscript was submitted to the to the Journal of Cell Science.

My role in the manuscript preparation was the conceptualization of the work, including formulation of the ideas and research goals, development of the methodology, management and coordination of the project, data presentation, and manuscript writing.

The experimental work was done by Dr. Zeynep Hein (immunofluorescence microscopy, Figure 4-1C, Figure 4-S8C), Swapnil S. Ghanwat (co-immunoprecipitation, Figure 4-2A), Dr. Benedict J. Chambers (NK cell killing assays, Figure 4-5D), and by myself (all other figures). Dr. Venkat R. Ramnarayan was involved in the formulation of the ideas and research goals, and writing of the original draft. Prof. Sebastian Springer supervised the project, acquired the funding, and supported the conceptualization and writing of the manuscript.

On 17 November 2020, the manuscript was published on the bioRxiv, a free on-line archive and distribution service for unpublished preprints in the life sciences. The content is available on-line using a Digital Object Identifier or a link:

DOI: https://doi.org/10.1101/2020.11.17.386763 https://www.biorxiv.org/content/10.1101/2020.11.17.386763v1

Title and authors

Title of the manuscript: The MCMV immunoevasin gp40/m152 inhibits NKG2D receptor activation by intracellular retention and cell surface masking of RAE-1g

Authors: Natalia Lis1, Zeynep Hein1¶, Swapnil S. Ghanwat1¶, Venkat Raman Ramnarayan1,2, Benedict J. Chambers3¶, and Sebastian Springer1

1 Department of Life Sciences and Chemistry, Jacobs University, Bremen, Germany;

2 Current address: Department of Molecular Biology, Universitätsmedizin Göttingen, Göttingen, Germany.

3 Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden

116 4. Manuscript

Summary gp40 (encoded by the m152 gene) is a murine cytomegalovirus immunoevasin that targets multiple types of immune response. Firstly, gp40 limits antigen presentation to cytotoxic T cells by retaining murine MHC class I molecules in the early secretory pathway (Janßen et al., 2016). Secondly, gp40 slows down Stimulator of interferon genes (STING)-mediated interferon response (Stempel et al., 2019). And third, gp40 inhibits NK cell activation by downregulating NKG2D-activating ligand RAE-1. The importance of gp40/RAE-1 interaction was recognized before, but the molecular and cellular mechanism of gp40-mediated RAE-1 trafficking retention has never been elucidated (Lodoen et al., 2003, Arapović et al., 2009).

In this chapter, I explain how gp40 downregulates the RAE-1 isoform RAE-1g using two separate mechanisms. First, using radiolabeling and re-immunoprecipitation I demonstrate that gp40 retains RAE-1g in the endoplasmic reticulum by tethering it to the host proteins of the p24 family, which carry retention and retrieval signals.

Second, I show that if the retention mechanism is saturated (which is likely to happen given the circumstances of viral infection), then gp40 still blocks the interaction of RAE-1g with the activating receptor on the NK cell, NKG2D, by masking RAE-1g at the cell surface. Cell surface staining with a recombinant NKG2D-Fc chimera confirms that gp40 and the NKG2D receptor compete for binding to RAE-1g. This finding, made in the live cell, concurs with the co-crystal structure solved the group of David Margulies (Zhi et al., 2010, Wang et al., 2012)

117 5. Additional projects and experiments

5 Additional projects and experiments

The majority of the results generated during my PhD thesis is included in the summitted manuscript [4]. In the following chapter, I describe additional experiments and results.

5.1 gp40 mutant that does not bind to RAE1g The aim of this project was to create a gp40 mutant that does not bind to RAE-1g. Such a mutant can be used as a control for the mass spectrometry analysis of the proteome co-immunprecipitated with gp40/RAE-1g complex, which was one of the aims at the beginning of my PhD project [1.10]. Based on the complex crystal structure, I identified the contact residues of gp40 and RAE-1g that form hydrogen bonds and have a major impact on the binding affinity (Table 5-1) (149).

Table 5-1. Residues of gp40 and RAE-1g crucial for the formation of a strong complex.

gp40 amino acid residues RAE-1g amino acid residues

Glu28 Trp21, Tyr22 Asp113 and Asn115 Arg73, Ser77, and Asn78 Asp236 Lys 154 Arg222 Glu159

I chose three amino acids of gp40, glutamic acid 28 (E28), aspartic acid 113 (D113), and aspartic acid 236 (D236), to mutate them to alanine. They are depicted in Figure 5-1A, and the responding RAE-1g amino acids are depicted in Figure 5-1B.

118 5. Additional projects and experiments

A B

Figure 5-1. Crystal structure of gp40 and RAE-1g. A) gp40 amino acid residues crucial for binding to RAE-1g, glutamic acid 28 (lime), aspartic acid 113 (cyan), aspartic acid 236 (blue). B) RAE-1g amino acid residues crucial for binding to gp40, tryptophan 21 (ice-blue), tyrosine 22 (lime), arginine 73 (orange), serine 77 (cyan), lysine 154 (yellow).

I obtained three different mutants containing between one to three and called E28A, E28A/D113A, and E28A/D113A/236A. The template used for the cloning was a C-terminally HA-tagged gp40WT, therefore all on the mutants also have an HA-tag.

I cloned gp40WT and newly obtained mutants in the GFP-expressing vector and transfected them into murine NIH3T3 fibroblast cells, which express endogenous RAE-1g. I measured the cell surface levels of RAE-1g by staining with CX1 (anti-RAE-1g monoclonal antibody). To test whether introduced mutations had an impact of mouse MHC class I molecules, I stained the cell surface with the 27-11-13s antibody against H-2Db. The single mutation E28A did not impact the gp40 effect on RAE-1g (data not shown). In contrast, introduction of two mutations, E28A and D113A, reduced downregulation of RAE-1g. Introduction of three mutations, E28A, D113A, and D236A, had a cumulative effect, and the majority of the cell surface RAE-1g was rescued. The downregulation of H-2Db was not influenced by introduced gp40 mutations (Figure 5-2).

119 5. Additional projects and experiments

A

B

Figure 5-2. RAE-1g and class I expression in cells co-expressing gp40 mutants A) NIH3T3 cells were transfected with an empty vector, gp40WT, gp40 E28A/D113A, or gp40 E28A/D113A/D236A. Cells were stained with the CX1 or 27-11-13s antibody. B) Normalized mean fluorescence intensity of the CX1 and 27-11-13s staining values normalized to the empty vector control.

120 5. Additional projects and experiments

I concluded that the gp40 E28A/D113A/236A fails to downregulate RAE-1g effectively, but the effect on MHC class I is unchanged, suggesting that the immunoevasin is correctly folded. To learn about maturation of the mutant, I expressed gp40WT or gp40 E28A/D113A/236A in the HEK293T cells, lysed the cells, treated the lysates with PNGaseF digest, and subjected to an SDS-PAGE analysis. Glycosylation patterns were comparable between the two types of gp40, suggesting that the mutant matures in the same way as the wild type protein (Figure 5-3). In conclusion, this mutant is a good control to study gp40WT/RAE-1g complex and its potential binding partners.

Empty vector gp40WT gp40 E28A/ D113A/ D236A PNGaseF: - + - + - + WB: gp40 55-

40-

35- 25-

WB: Calnexin 100-

Figure 5-3. Glycosylation of gp40 E28A/D113A/D236A. HEK293T cells were transfected with an empty vector, gp40WT or gp40 E28A/D113A/D236A. Cell lysates were subjected to the PNGaseF digest, resolved using SDS-PAGE, and stained with agp40 antibody. Calnexin stain was used as a gel-loading control.

Further characterization of gp40 E28A/D113A/D236A should include pulse-chase analysis of the trafficking, and a co-immuprecipitation experiment to confirm lack of binding to RAE-1g.

5.2 gp40 oligomerization Protein oligomerization has many potential advantages, for example, improvement of the function by accumulation of multiple active sites, introduction of new interaction surface and thus, new function, or allosteric regulation. For some proteins, oligomerization is a prerequisite of correct folding, while for others it enables multiple

121 5. Additional projects and experiments functions depending on the monomeric/oligomeric state (180). Popular methods used to study protein oligomerization are co-immunoprecipitation, gradient separation, gel filtration chromatography, and domain swapping mutations (180–182).

Homo- and hetero- oligomeric proteins are common among viruses, for example, the HCMV tegument heterodimer of UL48 and UL47 necessary for secondary envelopment of the capsids; the HCMV major capsid protein (MCP); and the Flavivirus protein NS1 involved in the RNA replication as a dimer, or secreted as an immune-modulating hexamer (181, 183, 184).

Immunoevasins, like HCMV US3 and US6, were also shown to oligomerize; however it remains unclear whether, or how the oligomerization impacts the protein function [1.6]. US3 forms oligomers that are more stable than US3/MHC class I complex, and can be detected in vivo and in vitro. Like in gp40, separate parts of US3 are dedicated to binding to MHC class I and to ER retention. Moreover, US3 does not have any known retention signal, and probably it binds to an unidentified ER-resident protein. It has been suggested that US3 oligomerization enhances its function by formation of dynamic clusters in which binding to MHC class I and the unidentified retention factor is amplified and secured (182). In US6, different parts of the protein are dedicated to TAP inhibition, TAP binding, and oligomerization. Most probably, oligomerization allows more efficient TAP binding (185).

To test if gp40 can form oligomers, I co-expressed HA-tagged gp40WT and GFP-tagged gp40WT in HEK293T cells. After cell lysis in mild conditions (1% digitonin), I co-immunoprecipitated the protein with an anti-HA tag antibody, resolved proteins with SDS-PAGE, and performed immunostaining with an anti-GFP antibody. Additionally, I performed analogical experiment with cells expressing HA-tagged gp40LM and GFP-tagged gp40LM.

Results suggest that gp40WT indeed forms oligomers (Figure 5-4A). I expected that gp40LM would not form oligomers, because the linker was predicted to be a helix, and protein oligomerization is often based on a coiled-coil interaction (177, 186) structure. Surprisingly, that was not true, and gp40LM also forms oligomers (Figure 5-4B). The result suggests that another part of gp40, such as the lumenal domain, the transmembrane domain, or the cytosolic tail, facilitate the oligomerization.

122 5. Additional projects and experiments

A B

GFP

gp40LM

HA HA + GFP MW MW [kDa] [kDa] gp40WTgp40WT HA HAgp40WT + gp40WT GFP GFP gp40LMgp40LM gp40LM 80 - 58 - 80 - 46 - 58 - 46 - 1. WB : aGFP 32 - 32 - 25 - 25 - 22 - 22 - IP: aHA 80 - 58 - 80 - 46 - 58 - 46 - 2. WB : aHA 32 - 32 - 25 - 25 - 22 - 22 -

Figure 5-4. Oligomerization of gp40. A) HEK293T cells were transfected with gp40WT-HA, gp40WT-HA and gp40WT-GFP, or gp40WT-GFP. In each case the equivalent amounts of gp40 DNA was used for transfection, and in samples transfected with one type of gp40 construct, an empty vector was co-transfected so that each sample was transfected with the same amount of DNA. Next, cells were lysed in 1% digitonin, and the proteins were immunoprecipitated from the lysate with an anti-HA antibody. Samples were digested with PNGaseF, separated by SDS PAGE, and immunoblotted against GFP. After distaining, the membranes were immunoblotted against an HA tag. B) HEK293T cells were transfected with gp40LM-HA, gp40LM-HA and gp40LM-GFP, or gp40LM-GFP. Next, samples were treated the same as in A.

Further experiments should be performed to learn what is the degree of gp40 oligomerization, what part of the protein forms the oligomer interface, and whether oligomerization is important for gp40 function. Oligomeric gp40 may have different properties than monomeric one. It can have further consequences in inhibitory function or binding to the p24 family members or to target proteins, MHC class I, RAE-1g, or STING (117,152,153).

5.3 RAE-1g membrane anchorage RAE-1g is embedded into the membrane with a GPI anchor [1.5.2]. This type of membrane attachment can influence the maturation and mobility of the protein, and

123 5. Additional projects and experiments it may contain an ER exit signal. Some GPI-anchored protein ectodomains form transient homodimers or clusters within lipid rafts. The GPI anchor may influence binding between gp40 and RAE-1g, or it might impact the formation of the complex with the ER-anchoring binding partner, TMED10 (95, 186).

The type of membrane anchorage sometimes plays a role in immune evasion [1.6.1.5]. The truncated version of the human NKG2DL MICA, called MICA*008, is a GPI-anchored variant that is resistant to UL142 (an immunoevasin that downregulates only full length MICA), but sensitive to US9 (an immunoevasin that downregulates only GPI-anchored MICA). Transfer of the truncated C-terminal part of MICA*008 to the full length allele MICA*004, or MICB (both normally attached with a transmembrane domain) made those proteins susceptible to US9. Moreover, exchange of the C-terminal truncated sequence to a GPI anchor attachment sequence of another protein, ULBP3, resulted in US9-resistant MICA*008 (136). This example suggests that the type of membrane attachment impacts protein properties in the context of the viral immune evasion.

In order to learn whether RAE-1g downregulation by gp40 depends on the type of RAE-1g anchorage, I designed two HA-tagged RAE-1g mutants; first, the soluble RAE-1g; and second, a chimera of RAE-1g with a membrane anchorage of the MHC class I molecule H-2Kb. In soluble HA-RAE-1g, the GPI-anchor attachment site and proximal serine amino acid residues in the pro-peptide sequence are exchanged to glycine residues. In the chimera between HA-RAE-1g and H-2Kb, the serine modified with the GPI-anchor is exchanged to three glycine residues separating the HA-RAE-1g and H-2Kb parts of the protein, followed by the two amino acid residues of cytosolic H-2Kb part, transmembrane domain, and cytosolic tail of H-2Kb.

The wild type and mutated sequences of HA-tagged RAE-1g are as follows:

Wild type HA-RAE-1g MAKAAVTKRHHFMIQKLLILLSYGYTNGLDYPYDVPDYAGGGDAHSLRCN LTIKAPTPADPLWYEAKCLVDEILILHLSNINKTMTSGDPGETANATEVG

ECLTQPVNDLCQKLRDKVSNTKVDTHKTNGYPHLQVTMIYPQSQGQTPSA

TWEFNISDSYFFTFYTENMSWRSANDESGVIMNKWNDDGDLVQRLKYFIP

ECRQKIDEFLKQSKEKPRSTSRSPSITQLTSTSPLPPPSHSTSKKGFISV

GLIFK

124 5. Additional projects and experiments

Soluble HA-RAE-1g MAKAAVTKRHHFMIQKLLILLSYGYTNGLDYPYDVPDYAGGGDAHSLRCN LTIKAPTPADPLWYEAKCLVDEILILHLSNINKTMTSGDPGETANATEVG

ECLTQPVNDLCQKLRDKVSNTKVDTHKTNGYPHLQVTMIYPQSQGQTPSA

TWEFNISDSYFFTFYTENMSWRSANDESGVIMNKWNDDGDLVQRLKYFIP

ECRQKIDEFLKQSKEKPRSTSRSPSITQLTSTSPLPPPGHGTGKKGFIGV

GLIFK

Chimera MAKAAVTKRHHFMIQKLLILLSYGYTNGLDYPYDVPDYAGGGDAHSLRCN LTIKAPTPADPLWYEAKCLVDEILILHLSNINKTMTSGDPGETANATEVG HA-RAE-1g/H-2Kb ECLTQPVNDLCQKLRDKVSNTKVDTHKTNGYPHLQVTMIYPQSQGQTPSA

TWEFNISDSYFFTFYTENMSWRSANDESGVIMNKWNDDGDLVQRLKYFIP

ECRQKIDEFLKQSKEKPRSTSRSPSITQLTSTSPLPPPGGGSMATVAVLV

VLGAAIVTGAVVAFVMKMRRRNTGGKGGDYALAPGSQTSDLSLPDCKVMV

HDPHSLA

Legend: Lumenal part of RAE-1g HA tag Glycine residues separator Lipidation site (attachment of the GPI anchor) Serine to glycine mutation H-2Kb transmembrane domain H-2Kb cytosolic tail

The cloning of both RAE-1g mutants was performed by Bersal Williams as a part of his BSc thesis project, and the process is precisely documented in the thesis entitled “Tagging and Validation of Key Players in the Interaction Between MCMV and Its Host”. Unfortunately, only cloning of the soluble RAE-1g was successful, and cloning of the HA-RAE-1g /H-2Kb awaits further attempt.

After obtaining of one or both RAE-1g mutants, they can be tested when co-expressed with gp40. Experiments analogous to those described in [Error! Reference source

125 5. Additional projects and experiments not found.], namely cell surface and intracellular immunostaining and flow cytometry, immunofluorescence microscopy, intracellular complex formation with gp40WT and gp40LM, intracellular trafficking, binding to the NKG2DR, and activation of NK cells, will allow us to learn about the impact of the RAE-1g anchorage on its interaction with gp40. Such experiments may also bring a valuable information about RAE-1g maturation, trafficking, and role of membrane anchorage outside the context of the MCMV immune evasion.

5.4 RAE-1g glycosylation According to the Uniprot database, RAE-1g has five putative N-linked glycosylation sites, and other RAE-1 isoforms have between four and five putative N-linked glycosylation sites (Uniprot entry numbers are listed below Figure 1-5, [1.7.2]). The previous studies of RAE-1 isoforms did not focus on their glycosylation. However, one of the first reports analyzing the gene and amino acid sequence suggested that RAE-1 also has three putative O-glycosylation sites (91). However, that information is not in agreement with the O-glycosylation site prediction tool NetOGlyc 4.0 Server.

My observations provide additional information about RAE-1g glycosylation. A pulse-chase experiment performed in HEK293T cells transfected with HA-RAE-1g demonstrates that after synthesis, five forms of RAE-1g are present in the cells, including one without any glycans, and four glycosylated ones (Figure 5-5, lane 1). This suggests that not all predicted N-linked glycosylation sites are glycosylated in all molecules. Moreover, there is a glycosylation present in the samples after 30 and 60 minutes of chase that is resistant to PNGaseF (Figure 5-5, lanes 6 and 9).

As my plan included quantification of the total protein amount at the end of the chase, my aim was to treat samples such as there is only one signal that corresponds to the deglycosylated amino acid chain. The PNGaseF resistant signal may be explained by the fact that RAE-1g is heavily glycosylated, and even after denaturation the heterogenous sialic acid modifications prevent complete digest by the enzyme. Alternatively, PNGaseF or buffer used in the experiment was not fully active, and therefore did not complete the digest. Both, the enzyme and the buffer are made in the laboratory of Prof. Springer, and I refer to them as the homemade PNGaseF and buffer.

126 5. Additional projects and experiments

Chase (min): 0 30 60 Enzyme digest: x E P x E P x E P Line number: 1 2 3 4 5 6 7 8 9

HA⎼RAE-1g -

Figure 5-5. Glycosylation of RAE-1g.

HEK293T cells were transfected with HA-RAE-1g, pulse-labeled for 10 minutes and chased up to 60 minutes. Cells were lysed in 1% Triton X-100 buffer, and the proteins were immunoprecipitated from the lysate with an anti-HA antibody. Samples were left untreated, or digested with E - EndoF1 (an enzyme that removes glycans from proteins that are located in the pre-medial Golgi compartments), or P – PNGaseF, an enzyme that removes all N-linked glycans form the protein irrespective of their intracellular location, as indicated, and separated by SDS-PAGE

In order to find sample treatment conditions, in which the total protein is deglycosylated, I expressed HA-RAE-1g in HEK293T cells, and analyzed using pulse chase experiment. To test if the homemade reagents are working as expected, I compared the commercial PNGaseF and commercial buffer, homemade PNGaseF and commercial buffer, and both homemade PNGaseF and buffer; and all reagent combinations were equally effective (Figure 5-6, lanes 3, 4 and 8, red dot). This suggests that homemade reagents digest RAE-1g glycans as efficiently as the commercial equivalents.

To learn if heavy glycosylation limits the access of PNGaseF to the RAE-1g glycan base, I tested sialidase, an enzyme that removes the sialic acid modifications. Combined treatment using both enzymes resulted in more consistent signal, with lower molecular weight than the PNGaseF-resistant signal (Figure 5-6, lane 9, green dot). This demonstrates that the degree of glycosylation may restrict complete digest by the PNGaseF. However, it also suggests that RAE-1g is subjected to another type of glycosylation, most probably O-linked glycosylation.

O-linked glycans are attached to the OH- groups of serine or threonine, and the modification takes place in the rough ER and cis-Golgi compartments, after N-glycosylation and folding. It usually occurs in the amino acid chain region reach in serine, threonine, and proline (188).

127 5. Additional projects and experiments

Next, I used a commercially available mixture of glycosidases (Protein Deglycosylation Mix), which contains PNGaseF, sialidases and O-glycosydases with different activities, and a buffer that is compatible with all of the components. This reagent successfully removed all of the RAE-1g glycosylation, allowing quantification of the total protein amount, and also indicating that RAE-1g is modified with O-linked glycans (Figure 5-6, lane 12; Error! Reference source not found., [Error! Reference source not found.]).

1 2 3 4 5 6 7 8 9 10 11 12

......

1 No digest 2 EndoH digest 3 PNGaseF digest with home made enzyme and buffer 4 PNGaseF digest with commercial enzyme and buffer 5 No digest 6 EndoH digest 7 Sialidase digest in sialidase-optimized buffer 8 PNGaseF digest with home made enzyme and commercial buffer 9 Sialidase + PNGaseF digest 10 EndoH digest 11 PNGaseF digest 12 Protein deglycosylation mix digest

Figure 5-6. Digest of RAE-1g using different treatment conditions. HEK293T cells were transfected with HA-RAE-1g, pulse-labeled for 10 minutes and chased for 60 minutes. Cells were lysed in 1% Triton X-100 buffer, and the proteins were immunoprecipitated from the lysate with an anti-HA antibody. Samples were treated as indicated below the gel pictures, and separated by SDS-PAGE. White dot, unspecific signal; red dot, PNGaseF resistant band; green dot, presumable O-linked glycan

In order to confirm that the PNGaseF-resistant signal corresponds to O-linked glycosylation of RAE-1g, a combination of PNGaseF and O-glycosidase should be

128 5. Additional projects and experiments tested. Alternatively, the SDS-PAGE membranes can be subjected to immunostaining of O-linked glycans to confirm the identity of the signal.

5.5 RAE-1g trafficking in TMED10-deletion cells Once I established that gp40-mediated retention of RAE-1g depends on TMED10 (Error! Reference source not found., [Error! Reference source not found.]), I tested how RAE-1g traffics in DTMED10 deletion cells, in the presence and absence of gp40. I transfected HEK293TDTMED10 cells with HA-RAE-1g alone, or together with gp40WT. I performed the pulse-chase and re-immunoprecipitation of HA-RAE-1g as described previously (Error! Reference source not found., [Error! Reference source not found.]).

The ER exit rate of HA-RAE-1g was similar in the presence and absence of gp40WT, and it partially resembled HA-RAE-1g maturation in wild type HEK293T cells (Error! Reference source not found., [Error! Reference source not found.]). However, the heterogenous EndoF1-resistant Golgi population was not prominent in both conditions (Figure 5-7).

Chase (min): 0 0 0 15 30 60 120 120 EndoH: + - + + + + + - PDM: ------+

RAE-1γ - - EndoH sensitive

gp40 - RAE-1γ - - EndoH sensitive

Figure 5-7. RAE-1g trafficking in TMED10-deletion cells. HEK293TDTMED10 cells were transfected with HA-RAE-1g alone, or with gp40, were pulse-labeled for 10 minutes and chased for the indicated times. Cells were lysed in 1% Triton X-100 buffer, and the proteins were immunoprecipitated from the lysate with an anti-HA antibody. Immunoprecipitates (excluding the first lane) were dissociated with denaturation buffer, and proteins were re-immunoprecipitated with an anti-HA antibody. Samples were digested with EndoF1 (an enzyme that removes glycans from proteins that are located in the pre-medial Golgi compartments) or protein deglycosylation mix (PDM) (an enzyme mix that removes all glycans form the protein irrespective of their intracellular location), as indicated, and separated by SDS-PAGE.

129 5. Additional projects and experiments

A comparison of the quantified EndoF1-sensitive signals between wild type and TMED10-deletion cells suggests that knockout of TMED10 slows down trafficking of HA-RAE-1g. Surprisingly, in HEK293TDTMED10 cells, the total protein amount at the end of the chase was the same in the absence and presence of gp40, and it was higher than in HEK293T wild type cells expressing HA-RAE-1g alone, or with gp40WT or gp40LM. (Figure 5-8). Chart Title

RAE-1! 1,2 * RAE-1! + gp40WT HEK293T 1,0 RAE-1! + gp40LM 0,8 RAE-1! HEK293TDTMED10 0,6 RAE-1! + gp40WT 0,4 Series9

sensitive signal sensitive 0,2 Series10 Normalized EndoH1 EndoH1 Normalized 0,0 0 15 30 45 60 75 90 105 120 135 [min]

Figure 5-8. Quantification of RAE-1g trafficking in TMED10-deletion cells. Pulse-chase gels were quantified using ImageJ. Each value of the EndoF1-sensitive signal was normalized to the signal of the sample after 0 minutes of chase that was re-immunoprecipitated and treated with EndoF1. The last data point indicated with an asterisk corresponds to the sample treated with the protein deglycosylation mix and normalized to the signal of the sample after 0 minutes of chase that was re-immunoprecipitated and treated with EndoF1.

Lack of the sialylated population of HA-RAE-1g, irrespective of gp40 presence, would be normally associated with lack of protein transport from the ER to Golgi. However, such a conclusion contradicts the observation that the EndoF1-sensitive population decreases over time, the protein does degrade, and eventually it reaches the cell surface (Error! Reference source not found., [Error! Reference source not found.]). Possibly, HA-RAE-1g glycosylation is altered in the TMED10-deletion cells. First of all, in these cells, the Golgi compartment is fragmented (our own unpublished observation), and the glycan modifying enzyme activity may be less efficient. Secondly, it cannot be excluded that gp40 limits some of RAE-1g glycosylation modifications at later time points of the chase. Such an effect combined with the changed Golgi morphology, and impaired activity of the Golgi enzymes, may have a cumulative negative effect on the glycosylation. Pulse chase and re-immunoprecipitation of HA-RAE-1g in HEK293T wild type cells and TMED10 deletion cells without any enzyme treatment may indicate whether such a scenario is

130 5. Additional projects and experiments possible. To test if besides the TMED10 deletion, there is also a gp40-specific effect, such an experiment would also include co-expression of HA-RAE-1g with a gp40 mutant that does not bind RAE-1g [5.1]. The increased stability of HA-RAE-1g may be a consequence of slower protein trafficking. Information about the cell surface arrival rate of RAE-1g would complete the pulse-chase data. GPI-anchored proteins can be removed from the cells surface using Phospholipase C (189). After such a treatment, one would be able to measure the dynamics of HA-RAE-1g re-appearance at the cell surface.

To sum up, knockout of TMED10 has a global impact of cell morphology and trafficking of GPI-anchored proteins, and obstructs observation of gp40-specific effect on RAE-1g. Therefore, in order to study gp40/RAE-1g interaction, gp40LM expressed in wild type cells is a superior model.

5.6 MCMV infection of mouse cells To learn whether during MCMV infection, the p24 family-based retention mechanism of gp40 may be saturated, I infected wild type K41 murine fibroblast cells with MCMV. The result described in the manuscript demonstrates that indeed some cohort of gp40 is not retained, it escapes the ER and reaches the surface of infected cells (Error! Reference source not found.,[Error! Reference source not found.]). In addition, cells were also stained for intracellular gp40, the intracellular infection marker IE1 (m123), cell surface MHC class I, and cell surface RAE-1g. The aim was to was to assess whether the infection was successful. The experiments were performed according to the suggestions of Prof. Martin Messerle from Hannover Medical School.

During cytomegalovirus infection, the cell morphology such as the area of the cytoplasm and the nucleus are changed, and the cell size increases (190). When I infected K41 cells with MCMV, the FSC and SSC parameters were changed upon infection, indicating an alteration in size and granularity. Among cells incubated with MCMV, I observed some with the morphology typical for fibroblasts (uninfected cells) or enlarged with granular structures in the cytoplasm (infected cells) and round cells that are smaller than fibroblasts, with a bright rim, probably representing an intermediate stage of morphology during infection (Figure 5-9A). Therefore, I used two gating strategies to measure each staining, focusing either on all cells, or on those with a slight decrease in size, and increase of granularity (Figure 5-9B). Also, on each

131 5. Additional projects and experiments day of the infection, I stained uninfected cells along with the infected ones using the same protocol and antibodies.

A B Gating 1 Gating 2 SSC

FCS

Figure 5-9. Morphology of MCMV-infected cells. A) K41 cells were infected with MCMV and incubated for 24 hours. The picture was taken with the light microscope and a mobile phone camera. B) Infected cells were collected and analyzed with the flow cytometry.

5.6.1 Intracellular gp40 Intracellular gp40 staining demonstrated that, in agreement with the literature, the immunoevasin is expressed already 24 hpi, and the expression increases up to 72 hpi (Figure 5-10A). Cells analyzed using gating strategy 2 had a higher gp40 signal, both at the surface and intracellular, suggesting that in these cells, the infection was well established (Figure 5-10A-B).

132 5. Additional projects and experiments

A Gating 1 Gating 2

72 CMV + 1.04 3.05 hpi CMV - 0.18 0.18 48 CMV + 0.36 1.51 hpi CMV - 0.35 0.35 24 CMV + 0.53 0.67 hpi CMV - 0.25 0.25

Intracellular gp40 B 72 CMV + 0.35 0.77 hpi CMV - 0.21 0.21 48 CMV + 0.24 0.55 hpi CMV - 0.13 0.13 24 CMV + 0.19 0.34 hpi CMV - 0.13 0.13

Cell surface gp40

Figure 5-10. gp40 intracellular expression and cell surface expression. K41 cells were infected with MCMV, and incubated for indicated time. After harvesting, cells were incubated with FcR blocking reagent, fixed and permeabilized (only A) and expression of gp40 was determined by staining with anti-gp40 antibody, followed by APC, and flow cytometry. The number above each histogram indicates the mean fluoresce intensity of the sample.

5.6.2 Intracellular IE1 Staining of the intracellular IE1 protein is a commonly used way to control if the infection occurs in cells co-incubated with the MCMV (as suggested by Prof. Martin Messerle). Similarly to gp40, IE1 was expressed as soon as 24 hpi, and the expression increased within the incubation time. Cells analyzed using gating strategy 2 had a higher expression level of IE1, confirming that it was the population of cells with well-established infection (Figure 5-11).

133 5. Additional projects and experiments

Gating 1 Gating 2

72 CMV + 0.84 3.02 hpi CMV - 0.14 0.14 48 CMV + 0.45 1.56 hpi CMV - 0.29 0.29 24 CMV + 0.40 0.66 hpi CMV - 0.18 0.18

Intracellular IE1

Figure 5-11. IE1 intracellular expression. K41 cells were infected with MCMV, and incubated for indicated time. After harvesting, cells were incubated with FcR blocking reagent, fixed and permeabilized, and expression of IE1 was determined by staining with anti-IE1 antibody, followed by APC, and flow cytometry. The number above each histogram indicates the mean fluoresce intensity of the sample.

5.6.3 Cell surface H-2Kb Cell surface staining of the MHC class I molecule H-2Kb indicated that the downregulation effect was heterogenous, it was the most efficient 24 hpi, and it decreased over time. After 72 hpi, surface MHC class I was recovered in the majority of the cells. Also, the difference in the downregulation efficiency observed using gating strategy 1 or 2 was minor (Figure 5-12). A co-staining of IE1 and H-2Kb would allow to estimate the rate of MHC class I downregulation in infected cells. Unfortunately, this experiment was not performed due lack of antibodies conjugated with fluorophores.

134 5. Additional projects and experiments

Gating 1 Gating 2

72 CMV + 7.00 7.54 hpi CMV - 8.94 8.94 48 CMV + 3.53 5.64 hpi CMV - 13.9 13.9 24 CMV + 5.24 6.23 hpi - CMV 21.6 21.6

Cell surface H-2Kb

Figure 5-12. H-2Kb cell surface expression. K41 cells were infected with MCMV, and incubated for indicated time. After harvesting, cells were incubated with FcR blocking reagent, fixed and permeabilized, and expression of H-2Kb was determined by staining with Y3 antibody, followed by APC, and flow cytometry. The number above each histogram indicates the mean fluoresce intensity of the sample.

5.6.4 Cell surface RAE-1g K41 cells express a small amount of endogenous RAE-1g on their surface, and CX1 surface staining indicates that during MCMV infection RAE-1g was efficiently downregulated. The effect was strong during the first 48 hpi, decreased at 72 hpi, was observed in all of the cells, and was more prominent using gating strategy 1 than gating strategy 2 (Figure 5-13). A co-staining of gp40 and RAE-1g would allow to estimate the ratio between the two proteins, and efficiency of gp40 downregulation. Unfortunately, this experiment was not performed due lack of antibodies conjugated with fluorophores.

135 5. Additional projects and experiments

Gating 1 Gating 2

72 CMV + 0.25 0.34 hpi CMV - 0.45 0.45 48 CMV + 0.12 0.22 hpi CMV - 0.43 0.43 24 CMV + 0.14 0.22 hpi - CMV 0.50 0.50

Cell surface RAE-1g

Figure 5-13. RAE-1g cell surface expression. K41 cells were infected with MCMV, and incubated for indicated time. After harvesting, cells were incubated with FcR blocking reagent, fixed and permeabilized, and expression of RAE-1g was determined by staining with CX1 antibody, and flow cytometry. The number above each histogram indicates the mean fluoresce intensity of the sample.

The infection experiment was performed four times; one time using an MOI 3, without the centrifugal enhancement; and three times using an MOI 0.5, and with the centrifugal enhancement protocol. The results were comparable, and therefore, the second method was used to repeat the results.

For the future investigation of MCMV infection, an MCMV lacking the gene of interest, in this case MCMV Dm152, would be a valuable control. Additionally, using antibodies conjugated with fluorophores would allow to co-stain infected cells and proteins of interest, and analyse the effects of infection more precisely.

136 6. Outlook

6 Outlook

This study has reached most of the project aims [1.10] and provided valuable information about the immune evasion strategy of gp40 and the RAE-1g protein. The main finding of this work is that gp40 uses two strategies to stop the interaction of the NKG2D receptor with its ligand.

First, gp40 retains RAE-1g in the early secretory pathway, and the retention itself is maintained by binding to the TMED10 protein. gp40 forms a ternary complex where on one hand it binds to RAE-1g with the lumenal MHC class I-like domain, and on the other hand, it binds to TMED10 with the long and unstructured linker part (153). Second, if the retention mechanism is fails, gp40 and RAE-1g reach the cell surface as a complex, and gp40 sterically blocks or masks the interaction of RAE-1g with the NKG2D ligand. Additionally, I have shown that the retention mechanism may be saturated, and then gp40 reaches the cell surface during the MCMV infection. This is the first time when such a dual strategy combined of intracellular retention and cell surface masking was shown for a cytomegalovirus immunoevasin.

In the contrast to previous findings of other labs, I successfully co-immunoprecipitated gp40 and RAE-1g complex from the cells. The complex turned out to be more stable than gp40/MHC class I complex, as it resisted lysis in 1% Triton X-100. However, there are still many unknowns that may be interesting to follow up. The comparison of the binding affinities of gp40 to all of its targets, i.e., MHC class I, RAE-1g, and STING, has never been done, and such an information would indicate the priority sequence of these interactions. It is especially intriguing how during the MCMV infection, gp40 divides itself between these three targets, and what is the timeline of these interactions. Do they happen simultaneously, or one after another? Does oligomerization of gp40 play a role in the immune evasion of those proteins? Are there other MCMV proteins that regulate the activity of gp40 during infection, and, for example, direct it to one of the targets at a particular time of infection?

Precise pulse-chase analyses have revealed additional information about maturation of RAE-1g and suggest that this protein is modified not only with N-linked glycans but also with O-linked glycans. However, this finding requires further confirmation. Interestingly, the trafficking of RAE-1g in the TMED10 knockout cells was altered, suggesting that even though RAE-1g and TMED10 do not co-immunoprecipitate,

137 6. Outlook

TMED10 has an indirect effect on RAE-1g maturation and stability. Perhaps the TMED10 knockout has a global negative impact on trafficking and maturation of GPI-anchored proteins, and further studies of membrane anchorage and glycosylation of RAE-1g would allow to understand this trafficking alteration.

A few more aspects of this project can be developed further. First of all, it is still unclear what the fate of retained gp40/RAE-1g complex is. Based on the previous work on gp40/MHC class I, one may speculate that the complex gets degraded in the lysosome (151). It remains unclear how the complex would reach the lysosome, and whether it would migrate via the cell surface. Longer pulse chase analysis, and lysosomal or proteosomal inhibition studies, would allow us to track the fate of the complex. Also, the endocytosis rate on the gp40/RAE-1g complex, or RAE-1g alone has never been studied, and that would inform us about the cell surface stability of both proteins.

I have shown that the retention of the gp40/RAE-1g complex depends on the interaction of gp40 with TMED10. The next step would be to test whether other p24 proteins also anchor gp40 and RAE-1g. Most probably they do, since TMED2, TMED9, and TMED5 were also shown to co-immunoprecipitate with gp40. Knockdowns of those p24 proteins resulted in a rescue of cell surface MHC class I, and perhaps it has an analogous effect on RAE-1g (153).

Finally, the intracellular binding of gp40/RAE-1g, trafficking of both proteins, and cell surface masking should be further confirmed using the MCMV infection model. This may allow to evaluate how significant are both effects, the intracellular retention and cell surface masking, in infected cells. Combination of experiments performed with ectopically expressed gp40, and using MCMV infection model may allow to fully understand how one immunoevasin can inhibit immune response in multiple ways.

138 7. List of journal publications, conference participations and awards

7 List of journal publications, conference participations and awards

Publication co-authorship:

Ramnarayan V.R., Janßen L., Hein Z., Lis N., Ghanwat S., and Springer S. The p24 family protein TMED10/Tmp21/p24δ1 anchors a viral protein in the endoplasmic reticulum that abolishes MHC class I surface expression. Cell Reports; 2018; DOI: 10.1016/j.celrep.2018.05.017.

Conference participations:

I presented my project in a form of a poster presentation during three scientific meetings:

- 68. Mosbacher Kolloquium – „Cell Organelles – Origin, Dynamics, Communication“; 30.03-1.04.2017; Mosbach, Germany. - 28th Annual meeting of the society for virology;14-17.03.2018; Würzburg, Germany - 16th Workshop “Cell Biology of Viral Infections’’ of the German Society of Virology (GfV); 08-10.11.2017; Schöntal, Germany.

Awards:

I was awarded an EMBO Short-Term Fellowship (number 7905) to carry out research in the lab of Dr. Benedict Chambers (Karolinska Institute, Solna, Sweden). The topic of the research project was "MCMV m152/gp40 sterically prevents the binding of NKG2D to RAE-1g". The funding had been granted for a total period of 45 days.

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