Viral immune evasion strategies Frank Momburg Division of Molecular Immunology German Cancer Research Center, Heidelberg

1. Viral immune evasion strategies Frank Momburg Division of Molecular Immunology German Cancer Research Center, Heidelberg f.momburg@dkfz.de

2. Viral immune evasion strategies 1. Viral evasion of MHC class I-mediated antigen presentation 2. Viral evasion of MHC class II-mediated antigen presentation 3. Viral evasion of natural killer cell activation 4. Viral interference of chemokine/cytokine action 5. Viral evasion of antibody responses and complement attack 6. Viral interference with apoptosis

3. Life styles and survival strategies of viruses „hit and run“ „hit and hide“ Small RNA virus e.g. human influenza virus Large DNA virus e.g. CMV • Interference with innate immunity, typically type I interferon • Blockade of host gene transcription • Antigenic hypervariability • Interference with adaptive immunity • Replication at immunoprivileged sites • Molecular latency • In most cases: elimination of the virus • Persistent infection

4. Cytomegalovirus genome (235 kbp, > 200 open reading frames) Hepatitis B virus Influenzavirus Adenovirus Vaccinia virus CMV

5. Antigenic drift and antigenic shift of influenza virus

6. Acute infection and latent phase (herpes simplex virus)

7. Viruses involved in immune escape Herpesviruses HSV1,2 Herpes simplex virus types 1, 2 VZV Varicella zoster virus HCMV Human cytomegalovirus MCMV Mouse cytomegalovirus EBV Epstein Barr virus BHV-4 Bovine herpesvirus 4 EHV Equine herpes virus PRV Pseudorabies virus HVS Herpesvirus saimiri HHV-6,7,8 Human herpesvirus types 6, 7, 8 MHV-68 Mouse 2-herpesvirus 68 Poxviruses Myxoma virus Cowpox virus Vaccinia virus Molluscum contagiosum virus Variola virus Papovaviruses BPV Bovine papilloma virus HPV Human papilloma virus SV40 Simian virus 40 Retroviruses HIV Human immunodeficiency virus SIV Simian immunodeficiency virus MuLV Murine leukemia virus Others Adenovirus Influenzavirus RSV Respiratory syncytial virus MV Measles virus HCV Hepatitis C virus HBV Hepatitis B virus

8. The innate and the adaptive immune system

9. Presentation of antigenic peptides by MHC class I molecules Cytotoxic T cell Cb Ca T cell receptor Vb Va a1 a2 CD8 coreceptor a3 b2m Antigen presenting cell

10. Role of MHC class I-mediated antigen presentation in antiviral immune responses • The antigen-specific T cell receptors of CD8+ cytotoxic T cells (CTL) recognize MHC class I molecules displayed on the surface of virus-infected cells or on professional antigen presenting cells (dendritic cells, macrophages, B cells). • These MHC-I molecules present peptide antigens that are cleaved out of endogenously synthesized viral proteins by proteasomes and other intracellular proteases. Alternatively, viral antigens can be cross-presented by professional APC after uptake of viral particles or of apoptotic, virus-infected cells.

11. Viral subversion of MHC class I-mediated antigen presentation 1. Interference with the biosynthesis of MHC-I molecules AdenovirusE1ATranscriptional inhibition (via decrease of NFB) of MHC-I heavy chain, TAP1/2, and LMP2/7 expression 2. Interference with antigen processing by proteasomes HCMVpp65Kinase (IE phase), inhibits generation of antigenic peptides from a 72 kDa transcription factor (important antigen in immediate early phase) EBVEBNA-1Contains a 239 residue Gly-Ala repeat that blocks proteasomal processing MuLVenv-p15ESingle amino acid change (K  R) in FMR subtypes  loss of CTL epitope through altered proteasomal cleavage site

12. Viral subversion of MHC class I-mediated antigen presentation 2. Interference with antigen processing by proteasomes HCMVpp65Kinase (IE phase), inhibits generation of antigenic peptides of a 72 kDa transcription factor (important antigen in immediate early phase) EBVEBNA-1Contains a 239 residue Gly-Ala repeat that blocks proteasomal processing MuLVenv-p15ESingle amino acid change (K  R) in FMR subtypes  loss of CTL epitope through altered proteasomal cleavage site

13. Viral subversion of MHC class I-mediated antigen presentation 3. Interference with peptide transport by TAP HSV1/2ICP47Cytosolic protein blocking peptide binding to TAP HCMVUS6ER protein that inhibits peptide translocation (inducing a conformational change that prevents ATP binding to TAP1) BHV/EHV/PRVUL49.5Inhibition of peptide translocation (inducing a transport-incompetent arrest and proteasomal degradation of TAP) HIV?Inhibition of TAP

14. Viral subversion of MHC class I-mediated antigen presentation 4. Interference with the heterotrimeric assembly of MHC-I molecules in the ER HCMVUS2, US11Dislocation of MHC-I through the Sec61p channel to the cytosol  proteasomal degradation HIVVpuDislocation from ER to cytosol  proteasomal degradation AdenovirusE3/19KRetains MHC-I in the ER, inhibits TAP/tapasin association HCMVUS3Retains MHC-I in the ER, inhibition of tapasin- dependent peptide loading MHV-68mK3Ubiquitination/degradation of TAP/tapasin associated MHC-I HPVE5Retains MHC-I in the Golgi complex

15. Viral factors interfering with the peptide loading complex and assembly of MHC class I molecules

16. Retrotranslocation of MHC class I molecules from the ER to the cytosol for proteasomal degradation

17. Viral subversion of MHC class I-mediated antigen presentation 5. Interference with the intracellular trafficking of MHC-I molecules MCMVm152/gp40Retains MHC-I in cis-Golgi network MCMVm04/gp34Blocks antigen presentation by MHC-I on the cell surface MCMVm06/gp48Targets MHC-I to late endosomes/lysosomes  degradation HPVE5Retains MHC-I in the Golgi complex HIVNefEnhanced endocytosis of MHC-I  sequestration in trans-Golgi network HHV-8(KSHV)K3, K5Enhanced endocytosis of HLA-A, B, (C, E), ubiquitination/degradation HHV-7U21Diversion of MHC-I molecules to lysosomes  degradation

18. Viral factors obstructing the intracellular trafficking of MHC class I molecules

19. Viral inhibition of MHC class I-mediated antigen presentation 6. Mutation of CTL epitopes Mutations in class I-binding peptides represent an important mechanism to prevent antiviral CTL responses, leading to loss of MHC-I binding, loss of CTL recognition, or antagonism of an existing CTL response. HIV, EBV, HBV, HCV, Influenza virus

20. Multiple immune evasion mechanisms employed by HIV

21. Role of MHC class II-mediated antigen presentation in antiviral immune responses • Viruses that enter cells through phagocytosis or receptor-mediated endocytosis, or that are enveloped in the trans-Golgi network/early endosome, can undergo degradation into antigenic peptides through the action of endosomal/lysosomal proteases. Such peptides are loaded onto MHC class II (MHC-II) molecules for presentation to CD4+ T cells. • MHC-II molecules are constitutively expressed only by B cells, macrophages, dendritic cells and endothelial cells, however, can be induced on a variety of other cell types by IFN- in the course of inflammtory responses. • Activated CD4+ T cells function as helper T cells assisting the maturation of CD8+ CTL and thus coordinate antiviral responses, or may possess CTL activity themselves (in humans).

22. MHC class II-mediated presentation of exogenous viral antigens

23. MHC class II-mediated presentation of endogenous viral antigens

24. Viral evasion of MHC class II-mediated antigen presentation 1. Interference with the biosynthesis of MHC-II molecules MCMVM27IFN-induced transcriptional upregulation of MHC-II expression inhibited (interference with Jak1/STAT pathway  CIITA) AdenovirusE1AIFN-induced transcriptional upregulation of MHC-II expression inhibited

25. Interferon pathways

26. Viral evasion of MHC class II-mediated antigen presentation 2. Interference with the intracellular trafficking of MHC-II molecules HCMVUS2Dislocation of MHC-II to cytosol  proteasomal degradation EBVBZLF2Association with HLA-DR(DP, DQ)  chains on the cell surface  inhibition of antigen presentation BPVE6Interaction with AP-1 adaptor protein  disturbs intracellular transport BPV/HPVE5Interaction with 16K subunit of the vacuolar ATPase  endosomal acidification and MHC-II processing 

27. Viral interference with MHC class II-mediated antigen presentation

28. Helper T cell Cb Ca T cell receptor Vb Va CD4 coreceptor a1 b1 MHC-II/peptide b2 a2 Antigen presenting cell Viral inhibition of MHC class II-mediated antigen presentation 3. Interference with the costimulatory molecule CD4 (T helper cells) HIV-1,-2, SIVNefEndocytosis of CD4 via AP-2 adaptor complex (endocytosis motif in Nef), transport to lysosomes via COPI association HIV-1VpuInduction of CD4 ubiquitination in the ER  dislocation to the cytosol  proteasomal degradation Myxoma virus?Internalization of CD4  lysosomal degradation

29. Molecular mechanisms employed by the HIV Nef protein

30. Role of NK cells in antiviral immune responses • Natural Killer (NK) cells are important effector components of the innate immune system that function in the initial defense against viruses via direct cellular cytotoxicity and through the production of inflammatory cytokines that promote the influx of CD8+ T cells. • For the control of certain viral infections in mice (RSV, MCMV), an early NK-mediated cytotoxicity and IFN- production plays an important role. In humans, a congenital lack of NK cells is associated with severe herpesvirus infections and low NK cell numbers correlate with more rapid progession towards AIDS in HIV-positive patients. • NK cells are preferentially activated in the presence of low amounts of MHC-I molecules ("missing self") since particular allelic variants of polymorphic MHC-I molecules trigger inhibitory NK receptors.

31. Activation and inhibition of NK cells

32. Viral evasion of NK cell activation 1. MHC-I homologs binding to inhibitory NK receptors MCMVm144Peptide-free MHC-I homolog, 2-microglobulin- associated, inhibitory NK receptor unknown MCMVm157Distant MHC-I homolog; binding to Ly49I inhibitory receptor in CMV-susceptible mouse strain, to Ly49H activating receptor in CMV-resistant strain HCMVUL18MHC-I homolog, 2-microglobulin and peptide- associated, binds to NK-inhibitory LIR-1/ILT-2 receptor

33. Viral evasion of NK cell lysis 2. Selective expression of NK-inhibitory MHC-I molecules HIV-1NefNo downregulation of NK-inhibitory HLA-C, -E, -G molecules HCMVUS2, US11No downregulation of NK-inhibitory HLA-C, -E, -G molecules HCMVUL40Encodes a leader sequence-derived peptide that is loaded onto inhibitory HLA-E molecules in a TAP-independent fashion 3. Downmodulation of costimulatory molecules HHV-8 K5Decreased surface expression and ubiquitination of costimulatory(KSHV)ICAM-1 and B7-2 molecules HCVE2Ligation of costimulatory CD81(TAPA-1) tretaspan molecule

34. Viral mechanisms for evading NK cells

35. TAP-independent NK cell inhibition by HLA-E and the HCMV UL40 protein

36. Viral evasion of NK cell lysis 4. Inhibition of NK-activating ligands HCMVUL16Intracellular retention of the NK cell activating NKG2D ligands MICB and ULBP1,2 (does not affect MICA and ULBP3) MCMVm152/gp40Inhibits surface expression of NK activating ligand H-60

37. Inhibition of NK cell activation by HCMV UL16

38. Role of cytokines and chemokines in antiviral responses • Cytokines and chemokines are secreted polypeptides that coordinate inflammation, cellular activation, proliferation, differentiation, and chemotaxis. • Cytokines and chemokines are immune mediators that are produced early upon virus infection. They induce and maintain innate as well as adaptive immune responses. Cytokines are responsible for flu-like symptoms such as myalgia, fever, headache and drowsiness which are common manifestations of acute virus infections. • Cytokines can be powerful antiviral mediators, allowing clearance of virus infection in the majority of cases. Double-stranded viral RNA  type-I interferons (IFN-/)  protein translation , cell proliferation , cellular RNases for viral RNA . • Pro-inflammatory cytokines are of particular importance and frequently targeted by viruses: IL-1, IL-12, TNF-, IFN-/, IFN-, and several chemokines that activate leukocyte migration.

39. Viral evasion of cytokine action 1. Interruption of cytokine production and maturation AdenovirusE1ABlocks IRF-3-induced transcription of IFN-/ HPVE6Blocks IRF-3-induced transcription of IFN-/ HCMV?Inhibition of transcription of MCP-1 chemokine Myxoma virusSERP-2Inhibition of IL-1 converting enzyme (ICE)  IL-1 Cowpox virusCrmA/Spi-2Inhibits several caspases, including ICE Vaccinia virusB13RInhibits several caspases, including ICE Measles virusHemagglutininBinding to CD46 (complement receptor/regulator for C3b/C4b) on infected macrophages/DC  IL-12 production  Th1 response (IFN- production by T and NK cells) 

40. Viral interference with chemokine action

41. Viral evasion of chemokine/cytokine action 2. Interference with the receipt of the chemokine/cytokine signal A. Viral chemokine receptor homologs HCMVUS28Functional CCR1 chemokine receptor (binds MCP-1, MIP-1, RANTES), co-receptor for HIV entry HCMVUL33CCR1 chemokine receptor, expressed in viral coat MCMVM33CCR1 homolog, role in salivary gland dissemination and replication HVS ECRF3Functional CXCR2 chemokine receptor HHV-8ORF74Constitutively active, agonist-independent CXCR2 receptor HHV-6/7?Downregulation of cellular CXCR4 chemokine receptor in infected CD4+ T cells  response to ligand SDF-1  B. Viral chemokine/cytokine homologs HHV-8vMIP-IIBroad spectrum CC-, CXC- and CX3C chemokine antagonist MCVMC-148PBroad spectrum CC- and CXC chemokine antagonist HHV-8vMIP-IMIP-1 homolog, CCR8 agonist, Th2 response  HHV-6U83MIP-1 homolog, CC chemokine agonist MCMVm131CC chemokine homolog, promotes virus dissemination HHV-8 vIL-6IL-6 homolog, increases angiogenesis and hematopoesis (role in Kaposi sarkoma, IL-6R+) EBVvIL-10IL-10 homolog, antagonizes Th1 responses MCVMC-51LSecreted, binds IL-18  NK activation, Th1 response C. Virus-encoded secreted cytokine receptors and chemokine-binding proteins Cowpox virusCrmB, CrmCSecreted TNF-R homologs, sequester TNF- and LT- Myxoma virusMT-2Secreted TNF-R homolog, sequesters and inhibits TNF- HCMVUL144TNF-R homolog, retained intracellularly Vaccinia virusB18-RSecreted type I IFN-R homolog, sequesters IFN- Vaccinia virusB8-RSecreted type II IFN-R homolog, sequesters IFN- Myxoma virusM-T7Secreted type II IFN-R homolog, sequesters IFN- Vaccinia virusB15-RSecreted IL-1-R homolog, sequesters IL-1 EBVBARF-1Secreted protein, sequesters CSF-1

42. Viral evasion of chemokine/cytokine action 3. Interference with interferon-mediated effector functions A. Inhibition of IFN-induced gene transcription AdenovirusE1ADepletion of STAT1 or p48  ISGF3  IFN-induced transcriptional activation  HPVE7Sequestering of IRF1 IFN-induced gene transcription  HCMV?Depletion of Jak1 kinase and p48  ISGF3  HHV-8vIRFK9IRF antagonist, competition with IFN-induced transcription B. Interference with IFN-induced cellular defence mechanisms EBVEBNA2IRF antagonist, competition with IFN-induced transcription HCVE2Inhibits phosphorylation of eIF2 by dsRNA-dependent protein kinase (PKR) HSV134.5Activates protein phosphorylase 1 to dephosphorylate eIF2 Vaccinia virusK3LeIF2 homolog, inhibits PKR AdenovirusVAI RNAViral RNA, inhibits PKR EBVEBER IViral RNA, inhibits PKR Vaccinia virusE3LSequesters ds-RNA, prevents PKR and 2’5’-OS activation HSV2'-5'-(A) analogInhibits 2'-5'-oligoadenylate synthase  RNaseL activity 

43. Viral evasion of antiviral cytokine effector functions

44. Role of antibodies and complement in antiviral reponses • Protective antibodies bind to virus surface structures and can block their interaction with cellular receptors. • Antibody-tagged (opsonized) viruses can be cleared from the circulation via IgG-Fc receptors expressed by phagocytes or by germinal center follicular dendritic cells ( viral spread possible). • Fc receptors (CD16) instruct NK cells to lyse antibody-coated virus-infected cells by antibody-dependent cellular cytotoxicity (ADCC). • IgM- and IgG-coated viruses can be neutralized by the classical complement pathway. • Viruses tagged with the complement component C3b (or C4b, C3bi) can be phagocytosed via the CR1 (CD35) complement receptor. • Viruses coated with C3b cleavage products (C3d, C3bi) can activate the CR2 (CD21) complement receptor of B cells. Follicular dendritic cells expressing CR1/CR2 complement receptors trap opsonized viruses and stimulate antibody production by germinal center B cells. • Phagocytosis of opsonized viruses elicits antigen processing, release of pro-inflammatory cytokines and T cell activation. Byproducts of the complement pathway (C3a, C4a, C5a) function as chemotactic peptides and amplify the inflammatory process.

45. Fc receptors

46. Viral evasion of antibody responses Viral Fc receptor homologs HSV-1gE-gIFcR homolog for monomeric or aggregated IgG, expressed on virus particles or infected cells, gE contains YXXL internalization motif for endocytosis of immune complexes inhibition of ADCC HSV-2gEFcR homolog MCMVFcr1FcR homolog PRVgE-gIFcR homolog VZVgE-gIFcR homolog MVNPLigates cellular inhibitory FcRII receptor (CD32) downregulation of antibody production by B cells

47. Evasion of antibody responses by viral Fc receptors

48. The complement pathways and their regulation

49. Viral subversion of complement responses 1. Enhanced factor I-mediated inactivation of C3b, C4b, or C3 convertases by viral proteins mimicking regulators of complement activation (RCA) HSV1/2gC-1/2CR1(CD35) homolog HVS ORF-4C4b-BP, CD46(MCP), CD55(DAF) homolog HHV-8CCPHC4b-BP, CD46(MCP), CD55(DAF) homolog Cowpox virusIMPC4b-BP homolog Vaccinia virusVCPC4b-BP homolog Variola virusSPICEC4b-BP homolog HCMV?upregulation of host cell CD46 and CD55 HIV?downregulation of host cell CR1/CR2, recruitment of factor H by gp41 and gp120 2. Inhibition of C9 polymerisation HVSHVS-CD59CD59(MIRL) homolog

50. Viral evasion of complement activation