Viruses and host defenses

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					   Viruses and host defenses

How the successful virus beats the body
Learning objectives

• Describe the role of interferons, natural killer cells, innate
  and adaptive immune system in recovery from virus
• Explain how various viruses evade host immune strategies
• Interpret data from experiments designed to determine
  virus evasion mechanisms
Host outcome

      • Recovery
      • Persistence
      • Rapid death
     What prevents most viruses from causing

• Skin
• Nonspecific innate defenses
   – Natural killer cells
   – Interferon
   – Complement
   – Apoptosis
• Acquired immunity
   – Antibodies
   – Cytotoxic T cells
   – Lymphokines
    How do we know the importance of each host
         defense mechanism in recovery?

•   Infect and measure responses over time
•   Use mutant animals/genetic defects in humans
•   Infect and block specific defenses
•   Do in vitro studies
Innate defenses: we survive most virus infections

                         skin: impenetrable barrier unless insect
                             vector or wound
                         • mucous membranes: live target
                         • inflammatory response: neutrophils
                         • complement
                         • macrophages
                              – HIV integration/destruction;
                              – Dengue virus immune
                                  enhanced uptake
                         • cytokines
NK cells
Overcoming NK cells

          • CMV makes MHC homolog
          • How does it prevent cytotoxic T
            cells from killing virus infected
Interferon: host proteins induced by virus in vertebrates that
interfere with viral replication (type 1)

                                     • bind to receptor on host cells
                                       (determines species specificity)
                                     • through signal transduction
                                       induces synthesis of antiviral
                                       proteins (AVPs) controlled by
                                       interferon-stimulated response
                                       elements (ISRE) which protect
                                       cell from viral replication
• What would happen if you
  microinject IF into a cell?
• What would happen in presence
  of Actinomycin D?
• Evidence for IF role in recovery
  from infection
   – Time
   – Place
   – Exogenous transfer
   – Loss
Antiviral effects of IF-induced AVPs

                   • Oligoadenylate synthetase
                     activated in presence of dsRNA
                       – ATP-----> oligoA up to 15
                       – oligoA activates RNAase
                         that cleaves host and viral
                   • Protein kinase (PKR) activated
                     in presence of dsRNA
                     phosphorylates eIF2 needed for
Host cell responses are not always protective

                       • Results of microarrays using
                         high and low virulence strains
                          – Red are upregulated
                          – Green are downregulated
                          – Black - no change
                       • Increased inflammatory,
                         apoptotic, oxidative damage

    • neutralization: antigenic shifts
      and drifts(variation)
    • complement mediated lysis:
      vaccinia makes homolog of
    • HSV makes receptor for a
      complement component
cell mediated immunity: requires viral protein displayed
                      in MHC1

                             • downregulates transport of
                               MHC to surface (Adenovirus;
                               herpesvirus; measles)
                             • Increases endocytosis of MHC
                             • Neurons express little MHC
                             • Mutation to new epitopes
                             • latency
cell apoptosis

        • Blocked by viral proteins which
          may bind to host proteins
          promoting apoptosis
        • Produce mimic proteins that
          inhibit apoptosis
Multiple stolen genes of KSHV - HHV8

                   • V-FLIP - FLICE inhibitory
                   • Decoy receptor
                   •   Bcl-2 - anti-apoptotic factor
                   •   complement binding protein
                   •   IL6-like cytokine
                   •   three chemokines
                   •   interferon regulatory factor
                   •   D-type cyclin
                   •   G-protein coupled receptor
           How to make a killer virus

• What characteristics should a biological weapon
• How can it be constructed?
• Ectromelia virus causes
• Recovery due to CTL
  death of infected cells via
  perforin pathway
  mousepox virus produces
  inhibitors of caspases
• Vaccinia virus does not
  inhibit caspases so they
  are killed by two
• Il4 skews immune
  response to ab production
  and shuts down perforin
Ebola strategies

        • Releases soluble glycoprotein
          (portion of transmembrane
        • Hypothesis: It may bind to a
          host cell and prevent its activity
        • How do you show binding to
          specific white blood cell?

        • Gel depletion
         sGP vs GP binding to neutrophils vs endothelial

                                                   GP pseudotyped


                               QuickTime™ and a
                     TIFF (Un compressed) decompressor
                        are neede d to see this picture.


                                                       Infection by GP
                                                       pseudotyped luciferase
Ebola and IF induced gene expression

                   • Northern blots
                   • Which genes are affected by
                   • How would you determine
                     which EZ gene is responsible?
                        The Many Roles of Nef

                                         • Hypothesis: Downregulation of
                                         • How do you show this?
                                         • What can cause MHC
           Qui ckTi me™ and a
TIFF (Uncompresse d) d eco mpressor
   are ne ede d to see thi s pi cture.
              CD4 vs MHC downregulation

• Nef genes from WT, nonprogressors, slow progressors and rapid
• What is advantage to virus of CD4 downregulation?
• How can you determine whether the same or different parts of the Nef
  protein are responsible for each activity?
Increases viral
by interacting with
signal transduction
and TCR
                                                  Nef binds to p53 - what is
                                                    affect on apoptosis?

                                                                        •   How would you
                                                                            determine which
                                                                            proteins Nef binds

                                                                        •   Nef also binds to
Nef binds to p53. (a) A GST-Nef fusion protein specifically                 several cellular
coprecipitates p53. Purified recombinant GST-Nef (lane1) and                signal transduction
GST (lane 6) alone were incubated with purified recombinant p53,            elements -What is
affinity purified with glutathione-Sepharose beads,                         role?
electrophoresed, and transferred to nitrocellulose. Purified p53
                                                                        •   How do you show
was electrophoresed and transferred to nitrocellulose as a control
(lane 7). Nitrocellulose membranes were then reacted with anti-
                                                                            affect on apoptosis?
p53 in Western blotting. For competition of the GST-Nef-p53
interaction by purified Nef protein, p53 was incubated with
purified Nef protein at 0.3- (lane 2), 3- (lane 3), 10- (lane 4), and
30-fold (lane 5) molar excess before reaction with GST-Nef and
processing as described above
MOLT-4 cells which had been electroporated with purified recombinant full-
length Nef or GST or which had been mock electroporated were either exposed to
a lethal dose of UV light (+) for approximately 30 s or were not exposed (-) and
then returned to culture for 12 h. Hirt DNA was extracted from each of the
samples and analyzed by gel electrophoresis for the presence of fragmented
The APOBEC enzyme family
deaminates specific cytidine (C)
residues in either DNA or mRNA,
converting them to uridine (U)
       • Viruses replicating in cells
         producing APOBEC3G need
         Vif gene for cells to be
       • Vif- can grow in cells without
Anti IF strategy of HCV

            • NS5a binds to PKR and
            • E2 gene has 12 aa homology to
              autophosphorylation site of
              PKR and eIF2a
            • How do IFres and Ifsens differ?
            • How might that help the virus?
Do PKR and E2 bind?

          • His tag binds to beads
          • Isolate and run on gel
          • Wt PKR
          • K296 = mutant in ATP binding
          • E2-C - no Phos site
          • Hn - cell protein control
Does E2 interfere with PKR activity?

                   • ATP- P32
                   • PKR +/- E2 and in presence of
                     dsRNA activator and substrate
       HSV blocks IF in several ways
         vhs degrades mRNA, ICP27 prevents splicing

          QuickTime™ and a
TIFF (Un compressed) decompressor
   are neede d to see this picture.

                                              Virus destroys
                                              Jak1, disperses
                                              ND10 and
                                              disrupts PML

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