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  • Viral Evasion Strategies

    1

    Lecture 14Virology W3310/4310

    Spring 2012

    Tuesday, March 6, 2012

  • Dont look back, something might be gaining on you!

    Satchel Paige

    2

    Tuesday, March 6, 2012

  • 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

  • Acute Infections

    SymptomsWhen to treat?

    5

    Tuesday, March 6, 2012

  • Virus Offense Meets Host Defense

    6

    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

    7

    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 neutrophils

    8

    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

    9

    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

    10

    Tuesday, March 6, 2012

  • Cytokines

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

    11

    Tuesday, March 6, 2012

  • Viral Response

    Virokines, it looks like, smells like, so dont 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

    Tuesday, March 6, 2012

  • Modulators of the IFN Response

    14

    Tuesday, March 6, 2012

  • 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

    15

    Tuesday, March 6, 2012

  • Dissolution of PML

    16

    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

    eIF2 kinases

    - inhibit translation

    - consequences for virus replication

    17

    Tuesday, March 6, 2012

  • Innate Defense Targets

    18

    Tuesday, March 6, 2012

  • PKR

    Activated by binding dsRNA Autophosphorylates at S51 Phosphorylates eIF2

    - 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 34.5 dephosphorylate eIF2

    - 34.5 interacts with PP1a redirecting it to eIF2

    20

    Tuesday, March 6, 2012

  • Phosphorylation of eIF2

    US11 a proteinAd VA RNAs

    HPV E6HSV 34.5

    21

    Tuesday, March 6, 2012

  • Viral Modulators of Interferon

    Inhibit IFN synthesis IFN Receptor decoys Inhibition of IFN signaling Block function of Interferon Stimulated

    Genes

    22

    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 aggregates1517. 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 genedeficient 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 acidtype 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

    Atg1