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
3
Tuesday, March 6, 2012
Strategies for Evasion
• Overwhelm the host
• Enter parenterally - Anyway other than the gut - Why?
• Disarm host defenses
4
Tuesday, March 6, 2012
Evasion
• Disarm innate immunity
• Regulate MHC molecules - responsible for antigen presentation
• Interfere with CTL and NK cells
• Alter antigen presentation
• Go and hide More on this later
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Tuesday, March 6, 2012
Host Defenses• Immune system, recognizes signal, amplifies
signal and controls invader
• Innate immunity, responds to everything
• Intranuclear modulatory molecules
• Interferons induce an antiviral state
• PKR
• Complement punches holes in membranes
• NK and CTLs
• Macrophages and neutrophils8
Tuesday, March 6, 2012
Adaptive Immunity
• A memory response
• Activated in response to the innate immune system
• Results in clonal expansion of B and T cells - requires Th1 and Th2 cells
• Generation of Cytotoxic T celLs and antibody production
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Tuesday, March 6, 2012
Inflammatory Response
• Occurs in response to necrosis
• Results in release of cytokines and chemokines
• Recruits neutrophils and macrophages to site of damage
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Cytokines
• Primary output of innate immune response
• Rapid induction in response to infection
• Control inflammation
• Induce antiviral state, IFNs
• Regulate immune system
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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
12
Tuesday, March 6, 2012
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
13
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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
• HSV ICP0 targets PML for proteolysis
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Tuesday, March 6, 2012
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
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Tuesday, March 6, 2012
PKR
• Activated by binding dsRNA
• Autophosphorylates at S51
• Phosphorylates eIF2α - forms a very tight ternary complex with GDP- eIF2B - blocks recycling - translation is arrested
19
Tuesday, March 6, 2012
How Viruses Counteract Pkr
• Virus proteins have evolved to thwart host anti-virus defenses
• Herpes simplex virus US11 blocks Pkr activation
• 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
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Tuesday, March 6, 2012
Viral Modulators of Interferon
• Inhibit IFN synthesis
• IFN Receptor decoys
• Inhibition of IFN signaling
• Block function of Interferon Stimulated Genes
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Tuesday, March 6, 2012
Autophagy
23
For example, the most commonly used autophagy assays, biochemical detection of the lipidated form of LC3 (LC3-II) and detec-tion of the localization of LC3-II to punctate dots by light microscopy, are subject to many interpretations. Most investigators now rec-ognize that detection of increased LC3-II can indicate either more autophagosome forma-tion or a block in autophagosomal matura-tion, which necessitates the use of ancillary approaches to distinguish between these possibilities14. Perhaps less well appreciated is the idea that LC3 dots may represent the targeting of LC3 to structures other than autophagosomes, such as phagosomes, dou-ble-membraned scaffolds for the replication complexes of positive-strand RNA viruses, or even protein aggregates15–17. Similarly, LC3 may become lipidated, forming LC3-II, in the absence of autophagosome formation18,19. Thus, whereas LC3-II formation and localiza-tion of LC3 punctae are hallmark features of autophagosome formation (and sensitive parameters for the detection of autophagy), they lack complete specificity as markers of classical autophagy. The direct demonstra-tion of a function for autophagosomes in a process is the gold standard for proving that autophagy is involved. An extensive discus-sion of the various assays used in autophagy research, as well as the criteria for their use and interpretation, has been provided in a consen-sus paper published by many of the authorities in the field14.
The genetic knockout or knockdown of core autophagy genes is an effective way to turn off the autophagy pathway, but it is often unclear whether resulting phenotypes are due to a deficiency of classical autophagy or autophagy-independent functions of the autophagy genes. As reviewed elsewhere, autophagy genes are known to have alternative functions, including those in other membrane-trafficking events, axonal elongation and cell death20,21. Furthermore, investigators often assume that phenotypes noted in autophagy gene–deficient cells or organisms are due to lack of classical autophagy without providing direct experi-mental proof. Notably, for many studies of the involvement of autophagy genes in immunity (Fig. 3), it is not clear how classical autophagy, involv-ing the autophagolysosomal degradation of sequestered contents, could contribute to the described functions of autophagy genes. Thus, it is possible that autophagy genes act in other cellular processes that affect immune effector cell development and function. Here we will discuss advances related to the function of autophagy genes in the immune system.
Autophagy genes in antimicrobial defenseMore than a decade ago, the first published paper on Beclin 1, a mam-malian autophagy protein, demonstrated an antiviral function for exogenous neuronal expression of Beclin 1 in mice with alphavirus encephalitis22. Subsequently, endogenous autophagy genes were shown to be critical for the successful innate immune response to fungal, bacte-rial and viral pathogens in plants23, and viral evasion of Beclin 1 function by a herpes simplex virus neurovirulence factor was found to be essential
for lethal encephalitis in mice24. These studies collectively suggested a likely function for autophagy genes in pathogen defense in vivo. In parallel, many studies demonstrated a function for autophagy in vitro in defense against invading pathogens, including group A Streptococcus, Shigella flexneri, Mycobacterium tuberculosis, Salmonella typhimurium and Toxoplasma gondii10,11,13.
Two recent studies have further confirmed an antimicrobial func-tion for autophagy genes in host defense in vivo against intracellular pathogens and have identified previously unknown relationships among innate immune signaling, autophagy genes and potential autophagy-independent functions of autophagy genes. An innate microbial sensor, the peptidoglycan-recognition protein PRGP-LE, which recognizes bac-terial diaminopimelic acid–type peptidoglycan, is crucial for autophagic control of Listeria monocytogenes infection in fly hemocytes and for host survival25. PRGP-LE-mediated resistance is associated with autophagy induction, requires the autophagy gene Atg5 and presumably involves the xenophagic degradation of bacteria (as bacteria are visualized in autophagosomes in wild-type animals). In this case, xenophagy tar-gets cytoplasmic bacteria that have escaped from the endosome for envelopment in an autophagosome and destruction. In other cases in which a pathogen inside a vesicular structure is targeted (such as mycobacteria inside phagosomes, or salmonella inside vacuoles), the membrane dynamics involved are incompletely defined. It may be that an autophagosome can envelop the entire vesicular structure containing
462 VOLUME 10 NUMBER 5 MAY 2009 NATURE IMMUNOLOGY
AUTOPHAGY
Vesiclenucleation
Isolationmembrane
Vesicleelongation
Vesicle breakdown& degradation
Autolysosome
Lysosome
Autophagosome
Docking &fusion
Atg5
Atg16L1
Atg16L1
Atg16L1
Atg16L1
Atg12
Atg16L1
LC3
Atg10 E2Atg5
Atg12
Atg5
Atg12
Atg5
Atg12
Atg5 Atg12
Atg5
Atg12
Atg4
LC3 -GlyAtg3 E2
LC3PE
LC3
Atg7 E1
Beclin 1
Class III PI(3)K complex
Vps34
Vps15Atg14
Bcl-2–Bcl-XL
Otherregulators
Ubiquitin-like conjugation systems
P
Autophagy suppression
Nutrient abundance
Insulin-Akt-TOR signaling
Immune signals:
IL-4
IL-13
FADD
Immunity-related GTPases
Autophagy induction
Starvation
Growth factor deprivation
Immune signals:
IFN-
TNF
TLRs
PKR-elF2 kinase
Jnk
FADD
Immunity-related GTPases
Figure 1 The autophagy pathway and its regulation. The autophagy pathway proceeds through a series of stages, including nucleation of the autophagic vesicle, elongation and closure of the autophagosome membrane to envelop cytoplasmic constituents, docking of the autophagosome with the lysosome, and degradation of the cytoplasmic material inside the autophagosome. Vesicle nucleation depends on a class III PI(3)K complex that contains various proteins (in light yellow box at right), as well as additional proteins that regulate the activity of this complex (such as rubicon, UVRAG, Ambra-1 and Bif-1). Vesicle elongation and completion involves the activity of two ubiquitin-like conjugation systems (light blue 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, phosphatidylethanolamine; Gly, glycine; E1 and E2, ligases for ubiquitin-like conjugation systems.
REV IE W
a catabolic process involving degradation of a cell's own components through the lysosomal machinery
Tuesday, March 6, 2012
25
Stimulation and Inhibition of Autophagasome Formation
Mock HCMV UV-HCMV
4 h
8 h
24 h
LC3 an autophagy marker
pp65 HCMV protein
ds RNA Binding Domain
PKR Binding Domain
TRS1 (1-795)
Beclin Binding Domain
TRS1 an Inhibitor
Tuesday, March 6, 2012
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
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Tuesday, March 6, 2012
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
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Extrinsic Death Receptor Pathway
Ligands
Receptors
Activation of Caspase 8
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Characteristics of Apoptosis
• Cell organelles are dismantled
• Vesicle formation and membrane blebbing
• DNA is cleaved
• Phosphatidylserine, annexin appear on cell surfaces
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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
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Characteristics of Apoptosis
• Membrane blebbing and apoptotic body formation
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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
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Tuesday, March 6, 2012
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)
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Tuesday, March 6, 2012
HIV Sequesters RIG-I to Lysosomes Where It Is Degraded
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GFP-Protease+
Saquinavir
GFP-Protease RIG-I LAMP-1 Merge
GFP-Protease
GFP RIG-I Cytoplasmic
RIG-I Perinuclear
RIG-I Perinuclear
Tuesday, March 6, 2012
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?
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Tuesday, March 6, 2012
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
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Tuesday, March 6, 2012
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
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Tuesday, March 6, 2012
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 cell43
Tuesday, March 6, 2012
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
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Tuesday, March 6, 2012
BCR and TCR
• Both bind antigen
• B cells as linear epitopes
• T cells as peptides
• Binding releases cytokines - stimulates cell division
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T Cell Function• CD4+ Th cells differentiate into Th1 and Th2 cells
46
Differentiation
Cross RegulationTh1 Th2IL-4IL-5IL-10
IL-2IL-12IFN-γ
Cell-mediated immunitydominates proinflammatory
response
Antibody responsedominates
Th1 vs Th2 responseAg-presenting cell
+Immature CD4+ Th cell } Lymph node
Immune cross-regulation by cytokinesTh1 response Th2 response
Enhance
IL-2IL-12IFN-γ
Suppress
IL-4IL-10
Enhance
IL-4IL-5IL-6IL-10
Suppress
IL-12IFN-γ
Tuesday, March 6, 2012
Two signal systems, MHC and accessory molecules and their ligands - lead to T cell activation
T Cell Surface Molecules and Ligands
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Tuesday, March 6, 2012
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
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Remediation of T Cell Response
• EBV encodes an IL 10 homologue that suppresses the Th1 response - thus sparing infected B cells
• KSHV elaborates a horde of virokines (cytokine mimetics) that provide a refuge from immune surveillance
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• 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
Natural Killer Cells
IR>>AR
50
I
Tuesday, March 6, 2012
Virus Modulation of NK Cells
• MHC I homologs
• Regulators of MHC I
• Release of virus-encoded cytokine-binding proteins (Viroceptors) block engagement of activating receptor
• Antagonist of activating receptor
• Infection of NK cell can lead to disruption of function or cell death
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Tuesday, March 6, 2012
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
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Tuesday, March 6, 2012
MHC I Presentation
55
MHC I
β2μ
cis
medial
trans
CD8
α chain
Tap1Tap 2
Calnexin
proteasome
T-cell receptor
β2μ
Lumen of ER
Ub-dependentproteolysis
Peptide in bindinggroove
Protein
Transportvesicles
Presenting cell
Golgi
Cytoplasm
T cell
Tuesday, March 6, 2012
Fine Structure of MHC I Presentation
56
• Viral proteins interfere with MHC I mediated antigen presentation
• Just how many targets are there?Tuesday, March 6, 2012
Intercession by HSV-1 ICP47
57
• Prevents binding of peptides to TAPICP 47 retained in the ER
Tuesday, March 6, 2012
Intercession by Ad E19
• Blocks MHC I -Tapasin interaction excludingit from the peptide loading complex
58
Tuesday, March 6, 2012
Intercession by HCMV US6
• A 23k Da ER-restricted glycoprotein with luminal and cytoplasmic domains that inhibits ATP binding to TAP thus preventing peptide loading
59
Tuesday, March 6, 2012
Intercession by EBV BNLF2a
• Prevents peptide and ATP binding to TAP like ICP47 and US6
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Tuesday, March 6, 2012
Intercession by HCMV US3
• A glycoprotein that interacts with Tapasin resulting in MHC 1 heavy chain degradation
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Tuesday, March 6, 2012
• Targets peptide-loading complex-incorporated MHC 1 for degradation by ubiquitination, interacts with both TAP and Tapasin
Intercession by CMV mK3
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Tuesday, March 6, 2012
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
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Tuesday, March 6, 2012
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
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• Secreted Modulators
• Modulators on infected cell surface
• Stealth
• Antigenic hypervariability
• Bypass or kill lympocytes
• Block adaptive immune response
• Inhibit complement
• Modulate apoptosis
• Modulate autophagy
• Interfere with pattern recognition receptors
Immune Modulation Strategies
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