Viral Evasion Strategies

Lecture 15 Virology W3310/4310 Spring 2013

“Don’t look back, something might be gaining on you!” -SATCHEL PAIGE Host vs. Virus

• What one does to the other? • Evolution of strategies to evade innate and adaptive cell responses to infection - goal: survival, reproduction and release

2 Strategies for Evasion

• Overwhelm the host • Enter parenterally - Anyway other than the gut - Why? • Disarm host defenses

3 Acute Infections

When to treat? Symptoms

4 Virus Offense Meets Host Defense

5 Evasion • VIPRS - viral proteins that interfere with antigen presentation • Disarm innate immunity • Regulate MHC molecules - responsible for antigen presentation • Alter antigen presentation • Interfere with CTL and NK cells • Go and hide More on this later 6 Host Defenses • Innate immunity, responds to everything • Intranuclear modulatory molecules • Immune system, recognizes signal, amplifies signal and controls invader • Interferons induce an antiviral state PKR • Complement punches holes in membranes • NK and CTLs • Macrophages and neutrophils 7 Adaptive Immunity

• A memory response • Activated in response to the innate immune system • Results in clonal expansion of B and T cells - requires CD4+ Th1 and Th2 cells • Generation of Cytotoxic T celLs and antibody production

8 Inflammatory Response

• Occurs in response to necrosis • Results in release of cytokines and chemokines • Recruits neutrophils and macrophages to site of damage

9 Cytokines

• Primary output of innate immune response • Rapid induction in response to infection • Control inflammation • Induce antiviral state, IFNs • Regulate immune system

10 Viral Response • Virokines, it looks like, smells like, so don’t step in it - these mimetics bind and sequester host receptor molecules • Viroceptors - soluble cytokine receptors - divert cytokines from initiating response • Sabotage innate and adaptive defense without affecting growth in cell culture

11 Products that Counter IFN • Why? - Without IFN host has a reduced ability to contain viral infections • dsRNA binding proteins - NS1 from Influenza - E3h from Poxviruses - US11 from HSV • Ad VA-RNA - A dsRNA decoy that binds PKR

12 Modulators of the IFN Response

13 Regulators of ISGs

• ND10s are composed of host proteins that repress virus replication - innate nuclear defense - epigenetic regulation of virus replication - PML, an important ND10 constituent • Some Herpesviruses targets PML for proteolysis

14 Dissolution of PML

15 Translational Regulation

• Viruses modify host to favor synthesis of their own proteins • IFNs establish an anti-viral state • Induction of Protein Kinase R and other eIF2α kinases - inhibit translation - consequences for virus replication

16 Innate Defense Targets

17 PKR

• Activated by binding dsRNA • Autophosphorylates at S51 • Phosphorylates eIF2α - forms a very tight ternary complex with GDP- eIF2B - blocks recycling - translation is arrested

18 How Viruses Counteract Pkr

• Virus proteins have evolved to thwart host anti- virus defenses • HSV US11 blocks Pkr activation by binding to it • Adenovirus VA RNAs bind tightly to Pkr - dsRNA decoy • HPV E6 and HSV γ34.5 dephosphorylate eIF2α - γ34.5 interacts with PP1a redirecting it to eIF2α

19 Phosphorylation of eIF2α

HSV US11 Ad VA RNAs

HPV E6 HSV γ34.5

20 Viral Modulators of Interferon

• Inhibit IFN synthesis • IFN Receptor decoys • Inhibition of IFN signaling • Inhibit Interferon Stimulated Genes

21 REVIEW Autophagy a catabolic process involving degradation of a cell's own For example, the most commonly used autophagy assays, biochemical detection of components through the lysosomal machinery the lipidated form of LC3 (LC3-II) and detec- tion of the localization of LC3-II to punctate AUTOPHAGY dots by light microscopy, are subject to many Class III PI(3)K complex Vesicle interpretations. Most investigators now rec- nucleation Other regulators ognize that detection of increased LC3-II can Autophagy induction Beclin 1 Atg14

Bcl-2–Bcl-XL P Vps34 Vps15 indicate either more autophagosome forma- Starvation Isolation tion or a block in autophagosomal matura- Growth factor deprivation membrane tion, which necessitates the use of ancillary Immune signals: IFN-G approaches to distinguish between these TNF 14 possibilities . Perhaps less well appreciated TLRs Ubiquitin-like conjugation systems PKR-elF2 kinase Vesicle A is the idea that LC3 dots may represent the elongation Atg16L1 Atg12 Atg12 Jnk Atg10 E2 Atg12 Atg5 Atg5 Atg5 targeting of LC3 to structures other than Atg5 Atg16L1 FADD Atg16L 1 Atg12 Atg16L1Atg16L1 autophagosomes, such as phagosomes, dou- Immunity-related GTPases Atg5 Atg5

Atg7 E1 Atg12 Atg12 ble-membraned scaffolds for the replication PE LC3 -Gly complexes of positive-strand RNA viruses, or Autophagy suppression LC3 LC3 15–17 Atg3 E2 even protein aggregates . Similarly, LC3 Nutrient abundance Atg4 LC3 may become lipidated, forming LC3-II, in Insulin-Akt-TOR signaling the absence of autophagosome formation18,19. Immune signals: IL-4 Thus, whereas LC3-II formation and localiza- IL-13 Docking & Vesicle breakdown fusion & degradation tion of LC3 punctae are hallmark features of FADD autophagosome formation (and sensitive Immunity-related GTPases Autophagosome Autolysosome parameters for the detection of autophagy), Lysosome they lack complete specificity as markers of classical autophagy. The direct demonstra- tion of a function for autophagosomes in a 22 process is the gold standard for proving that Figure 1 The autophagy pathway and its regulation. The autophagy pathway proceeds through a series autophagy is involved. An extensive discus- of stages, including nucleation of the autophagic vesicle, elongation and closure of the autophagosome sion of the various assays used in autophagy membrane to envelop cytoplasmic constituents, docking of the autophagosome with the lysosome, and research, as well as the criteria for their use and degradation of the cytoplasmic material inside the autophagosome. Vesicle nucleation depends on a interpretation, has been provided in a consen- class III PI(3)K complex that contains various proteins (in light yellow box at right), as well as additional sus paper published by many of the authorities proteins that regulate the activity of this complex (such as rubicon, UVRAG, Ambra-1 and Bif-1). Vesicle in the field14. elongation and completion involves the activity of two ubiquitin-like conjugation systems (light blue The genetic knockout or knockdown of box at right). The autophagy pathway is positively regulated (green box at left) and negatively regulated (red box at left) by diverse environmental and immunological signals. TNF, tumor necrosis factor; PE, core autophagy genes is an effective way to phosphatidylethanolamine; Gly, glycine; E1 and E2, ligases for ubiquitin-like conjugation systems. turn off the autophagy pathway, but it is often unclear whether resulting phenotypes are due to a deficiency of classical for lethal encephalitis in mice24. These studies collectively suggested autophagy or autophagy-independent functions of the autophagy genes. a likely function for autophagy genes in pathogen defense in vivo. In As reviewed elsewhere, autophagy genes are known to have alternative parallel, many studies demonstrated a function for autophagy in vitro functions, including those in other membrane-trafficking events, axonal in defense against invading pathogens, including group A Streptococcus, elongation and cell death20,21. Furthermore, investigators often assume Shigella flexneri, Mycobacterium tuberculosis, Salmonella typhimurium that phenotypes noted in autophagy gene–deficient cells or organisms and Toxoplasma gondii10,11,13. are due to lack of classical autophagy without providing direct experi- Two recent studies have further confirmed an antimicrobial func- mental proof. Notably, for many studies of the involvement of autophagy tion for autophagy genes in host defense in vivo against intracellular genes in immunity (Fig. 3), it is not clear how classical autophagy, involv- pathogens and have identified previously unknown relationships among ing the autophagolysosomal degradation of sequestered contents, could innate immune signaling, autophagy genes and potential autophagy- contribute to the described functions of autophagy genes. Thus, it is independent functions of autophagy genes. An innate microbial sensor, possible that autophagy genes act in other cellular processes that affect the peptidoglycan-recognition protein PRGP-LE, which recognizes bac- immune effector cell development and function. Here we will discuss terial diaminopimelic acid–type peptidoglycan, is crucial for autophagic advances related to the function of autophagy genes in the immune control of Listeria monocytogenes infection in fly hemocytes and for host system. survival25. PRGP-LE-mediated resistance is associated with autophagy induction, requires the autophagy gene Atg5 and presumably involves Autophagy genes in antimicrobial defense the xenophagic degradation of bacteria (as bacteria are visualized in More than a decade ago, the first published paper on Beclin 1, a mam- autophagosomes in wild-type animals). In this case, xenophagy tar- malian autophagy protein, demonstrated an antiviral function for gets cytoplasmic bacteria that have escaped from the endosome for exogenous neuronal expression of Beclin 1 in mice with alphavirus envelopment in an autophagosome and destruction. In other cases encephalitis22. Subsequently, endogenous autophagy genes were shown in which a pathogen inside a vesicular structure is targeted (such as to be critical for the successful innate immune response to fungal, bacte- mycobacteria inside phagosomes, or salmonella inside vacuoles), the rial and viral pathogens in plants23, and viral evasion of Beclin 1 function membrane dynamics involved are incompletely defined. It may be that by a herpes simplex virus neurovirulence factor was found to be essential an autophagosome can envelop the entire vesicular structure containing

462 VOLUME 10 NUMBER 5 MAY 2009 NATURE IMMUNOLOGY 23 Stimulation and Inhibition of Autophagosome Formation Mock HCMV UV-HCMV

LC3 an autophagy 4 h marker

8 h

24 h

pp65 HCMV protein 24 How Does HCMV TRS1 Work? TRS1 an Inhibitor ds RNA Binding Domain PKR Binding Domain

TRS1 (1-795)

Beclin Binding Domain

• TRS1 Binds PKR and ds RNA Innate immune response is inhibited • Interacts with and sequesters Beclin Inhibits autophagy

25 Apoptosis

• Host escape mechanism leading to cell death and inflammation by cysteine proteases (Caspases) - induction of cytokines - infected cells release proteins that are subsequently presented by MHCII - activation of CTLs

26 Apoptosis • Catabolic process involving degradation of a cell's own components through the lysosomal machinery • Host controls induction and suppression • Antiapoptotic: BCL-2 family members block mitochondrial translocation • Proapoptotic: BAX and BAD cause induction of a caspase cascade by release of mitochondrial cytochrome C

27 Characteristics of Apoptosis

• Cell organelles are dismantled • Vesicle formation and membrane blebbing • DNA is cleaved • Phosphatidylserine, annexin appear on cell surfaces

28 Apoptosis

• Perturb the cell cycle and apoptosis is activated - cell falls apart and virus fails to complete replication cycle • Block it and virus can complete replication

29 Characteristics of Apoptosis

• Membrane blebbing and apoptotic body formation

30 Characteristics of Apoptosis

• HSV ICP27 prevents DNA fragmentation

31 Why Block Apoptosis?

• Why are cells induced after infection? - activation of quiescent cell machinery - checkpoint controls respond • Virus responds to complete replication - failure leads to decreased yields • Inhibit release of virus Antigens - eliminate T cell activation - evade immune response

32 How to Block It

• HCMV transcribes a noncoding RNA (β2.7) that binds a mitochondrial protein that triggers apoptosis • AD E1B binds BAX preventing caspase activation (intrinsic) • AD E3 blocks Fas (Death Receptor) - induced apoptosis (extrinsic)

33 HIV Evades the Rig I Innate Immune Response

34 HIV Sequesters RIG-I to Lysosomes Where It Is Degraded

GFP-Protease RIG-I LAMP-1 Merge

GFP RIG-I Cytoplasmic

GFP-Protease RIG-I Perinuclear

GFP-Protease + RIG-I Perinuclear Saquinavir

35 Apobec

• A protein family whose function is to edit RNA - an ISG - deaminates C U • Intrinsic antiviral • Blocks replication of HIV, HBV and Measles • Incorporated into HIV virus particles - Is the host a step ahead here?

36 How Apobec Inhibits HIV Replication

• C’s in - strand DNA are deaminated • C U transitions result in GC AT pairs - TGG (W) codons become TAA (Stop) • U containing DNA is attacked by U-DNA glycosidase - generates abasic site and becomes a target for endonucleases

37 How HIV Survives Apobec

• HIV encodes Vif which is incorporated in the virion • Vif interacts in a species specific manner to bind Apobec and target it to the proteasome 38 Humoral & Cell Immunity

MHC II MHC I

39 T Cell Surface Molecules and Ligands

Two signal systems, MHC and accessory molecules and their ligands - lead to T cell activation

40 41 Th2 Th1

42 Effects of Stimulating Cell Division • Clonal expansion - increases # of cell responders • T cells kill cells bearing foreign peptide or protein - CD4+ Th recognize MHC II bound peptides - CD8+ Tc recognize MHC I bound peptides • In response to cytokines from Th1 cells CD8+ become mature CTLs • In response to cytokines from Th2 cells B cell synthesizes Ab (becomes a plasma cell) • B cell can also become a memory cell 43 Adaptive Immunity

• Humoral • Cell-mediated, recognition mediated by: - membrane bound Ab on B cells and/or TCR - Ag presented by MHC I on all cells - Ag presented by MHC II on macrophages or dendritic cells

44 Immune Modulation

• Dendritic cells are often compromised by virus infection resulting in suppression of the immune response by: - interference with recruitment - impairment of Ag uptake or processing - interference with maturation - inability to migrate to lymphoid tissue - failure to activate T cells

45 Remediation of T Cell Response

• EBV encodes an IL 10 homologue that suppresses the Th1 response • KSHV elaborates a horde of virokines (cytokine mimetics) that provide a refuge from immune surveillance

46 Natural Killer Cells

I IR>>AR • No antigen receptors • Secrete cytokines • Kill cells lacking MHC I on the surface by recognizing missing self • ADCC, NKs bind to IgG coated cells, release perforins and granzymes triggering apoptosis 47 Virus Modulation of NK Cells

1-Mimetic

2-Downregulation & substitution

3-Block activating proteins

4-Inhibition of NK-stimulating cytokines or antagonist of activating chemokines

antagonist

5-Block receptor or kill cell

48 Virus Modulation of NK Cells

• MHC I homologs • Regulators of MHC I • Release of Viroceptors block engagement of activating receptor • Antagonist (Virokine) of activating receptor • Infection of NK cell can lead to disruption of function or cell death

49 Virus Modulation of NK Cells

• HCV E2 protein binds CD81 on NK cells to block activation - don’t recognize infected cells • HIV nef affects cell surface expression of some MHC I molecules but not HLA-E (cell is still protected) • Poxviruses express proteins that bind IL-12 to inhibit IFN-γ production by NKs

50 Exogenous Antigen Presentation

post-Golgi vesicle

51 MHC I Presentation

52 Fine Structure of MHC I Presentation

• Viral proteins interfere with MHC I mediated antigen presentation Just how many targets are there? • 53 Intercession of TAP Transport

HSV HCMV EBV

X peptide-binding X ATP-binding X ATP & Peptide-binding

54 Intercession with Tapasin

Adeno HCMV

X Blocks MHC I Tapasin X Interacts with Interaction Preventing Tapasin Resulting in Peptide Loading MHC I Degradation 55 Cytomegaloviruses a Paradigm for Interference with MHC • UL83 tegument protein inhibits proteasome processing by P* • US6 inhibits MHC I translocation to the ER • US3 inhibits MHC I transport across ER • US2 & 11 force MHC I to cytoplasm • UL18 is a MHC I mimetic thought to downregulate NK and CTLs

56 MHC Intercession Summary

• Viruses have to cope with their hosts • For the host to succeed viral antigens must be presented on the cell surface • Viruses have identified many points of intercession and defined how MHC I presents antigen

57 Immune Modulation Strategies • Secreted Modulators • Modulators on infected cell surface • Stealth • Antigenic hypervariability • Bypass or kill lymphocytes • Block adaptive immune response • Inhibit complement • Modulate apoptosis • Modulate autophagy • Interfere with pattern recognition receptors 58