Download Viruses and host defenses

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
infection
• 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
disease?
• 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?
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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
cells
• 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
cell?
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
experiments
– Loss
Antiviral effects of IF-induced AVPs
• Oligoadenylate synthetase
activated in presence of dsRNA
– ATP-----> oligoA up to 15
residues
– oligoA activates RNAase
that cleaves host and viral
mRNA
• Protein kinase (PKR) activated
in presence of dsRNA
phosphorylates eIF2 needed for
translation
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
responses
antibody
• neutralization: antigenic shifts
and drifts(variation)
• complement mediated lysis:
vaccinia makes homolog of
inhibitor
• 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
protein
• Decoy receptor
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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
have?
• How can it be constructed?
• Ectromelia virus causes
mousepox
• 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
mechanisms
• Il4 skews immune
response to ab production
and shuts down perforin
pathway
Ebola strategies
• Releases soluble glycoprotein
(portion of transmembrane
protein)
• 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
cells
GP pseudotyped
virus
neutrophils
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
endothelials
Infection by GP
pseudotyped luciferase
virus
Ebola and IF induced gene expression
• Northern blots
• Which genes are affected by
EZ?
• How would you determine
which EZ gene is responsible?
The Many Roles of Nef
QuickTime™ and a
TIFF (Uncomp resse d) d eco mpres sor
are nee ded to s ee this picture .
• Hypothesis: Downregulation of
MHC1
• How do you show this?
• What can cause MHC
downregulation?
CD4 vs MHC downregulation
• Nef genes from WT, nonprogressors, slow progressors and rapid
progressors
• 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
replication/budding
by interacting with
signal transduction
and TCR
Nef binds to p53 - what is
affect on apoptosis?
Nef binds to p53. (a) A GST-Nef fusion protein specifically
coprecipitates p53. Purified recombinant GST-Nef (lane1) and
GST (lane 6) alone were incubated with purified recombinant p53,
affinity purified with glutathione-Sepharose beads,
electrophoresed, and transferred to nitrocellulose. Purified p53
was electrophoresed and transferred to nitrocellulose as a control
(lane 7). Nitrocellulose membranes were then reacted with antip53 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
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How would you
determine which
proteins Nef binds
to?
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Nef also binds to
several cellular
signal transduction
elements -What is
role?
How do you show
affect on apoptosis?
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MOLT-4 cells which had been electroporated with purified recombinant fulllength 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
DNA.
The APOBEC enzyme family
deaminates specific cytidine (C)
residues in either DNA or mRNA,
converting them to uridine (U)
residues
• Viruses replicating in cells
producing APOBEC3G need
Vif gene for cells to be
permissive
• Vif- can grow in cells without
APOBEC
Anti IF strategy of HCV
• NS5a binds to PKR and
inactivates
• 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?
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His tag binds to beads
Isolate and run on gel
Wt PKR
K296 = mutant in ATP binding
domain
• 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
H2a
HSV blocks IF in several ways
vhs degrades mRNA, ICP27 prevents splicing
Virus
dephosphorylates
eIF2a
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Virus destroys
Jak1, disperses
ND10 and
disrupts PML