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MGY378 tt3 Exam Latest Update
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MGY378 tt3 Exam LatestUpdate
non-enveloped virus release: autophagy - Answer normal process in mammals
1) cytoplasm portion is enclosed by isolation memb, forming double membrane
structure = autophagosome
2) LC3B protein is recruited to autophagosome surface, and then P62 attaches to LC3B
in the inside of the autophagosome
4) autophagosome fuses w/ lysosome to degrade inner membrane, and then internal
contents = autolysosome
5) LC3B dissociates (reused for more autophagy), P62 degraded inside, AA and FA
released for recycling
cargo:
-self nourishment and metabolic recycling
-cellular homeostasis and elimination of unwanted/excess materials
non-enveloped virus release: experiment of nonlytic viral spread by autophagy - Answer
-poliovirus in hepatocyte huh cells
-used siRNA to knockdown LC3A and LC3B (important autophagosome protein) ->
injected viruses into cell (only a small amount to analyze SPREAD)
-mock: after 24h, clusters of red infected cells
-siRNA: infected cell cluster is smaller = less spread
-introduced loperamide (induces autophagy): in mock, signif bigger infected cell
clusters, but in siRNA, much smaller clusters and loperamide made no difference since
no LC3
-can virus spread without lysing/before infected cell is lysed? = at 10 hours, can see red
staining (cell infected) but no sytox staining (stains blue when cell is dead), showing that
donor cell is still intact but virus can spread w/o lysis (agar overlay to ensure close
contact spread)
-cells are damaged at a LATER time (sytox) than infected cell appearance
-donor/target PAIRS: in most times, 1st cell is blue before 2nd cell is red (pos values) but
w/ loperamide and nicardipine autophagy inducers, more cells release virus BEFORE
,dmg (neg values)
non-enveloped virus release: experiment of free virions vs vesicle virions - Answer
-poliovirus in heLa cells: infection w/ free virus vs virus in vesicles
-fluorescent in situ hybridization (FISH) to look at incoming viral genome from infected
virion
-if free virion, enter in diff cell areas but if in vesicle, arrives as a package =
complementation and recombination occurs, more fit genomes!
non-enveloped virus release: enterovirus transmission by autophagy - Answer 1) virus
enclosed by membrane = autophagosome formation
2) autophagosome goes to cell surface, outer membrane fuses and releases
extracellular vesicle
3) vesicle binds another cell thru PS receptor (phosphatidylserine) w/
phosphatidylserine on surface (which is also anti-inflammatory! = gives virus replication
advantage)
4) virus taken up in endosome
5) lipases in endosome degrades vesicle
6) the NORMAL VIRUS RECEPTORS are present in endosome = virions interact w/ own
receptor (required)
7) recombination, complementation of genome
non-enveloped virus release: how enteroviruses escape degradation in autophagy -
Answer -autophagosome: expresses STX17 on surface
-lysosome: expresses VAMP8 on surface
-SNAP29 connects STX17, VAMP8
-viral protease 3C cleaves SNAP29 = the two can't fuse
non-enveloped virus release: microvesicle pathway - Answer ex) rotavirus
-vesicles are derived from plasma membrane
non-enveloped virus release: exosome pathway - Answer ex) hep A/E, norovirus
-vesicles are derived from multivesicular bodies (vesicles w/ vesicles inside them)
-vesicles are more like exosomes
interferons: PKR pathway - Answer -PKR induced by type I IFNs in neighbouring cell ->
neighbouring cell must be activated by virus b/c pathway is activated by dsRNA ->
, PKR-P (autophosphorylation) -> phosphorylates eIF2a -> downregulates protein
synthesis
(RNA source can even be from DNA virus b/c parts of RNA in the virus can activate the
pathway)
viruses encode proteins that:
-downregulate PKR synthesis, degrade PKR, inhibit PKR, inhibit dsRNA, inhibit PKR-P
activity
-ex) adenovirus VA-RNA: binds to PKR, preventing its activation
ex) influenza NS1: binds to PKR, preventing activation and blocking induction of PKR
(diff mechanisms)
ex) HSV: dephosphorylates eIF2a, restoring protein synthesis
interferons: OAS pathway - Answer 2'-5' A synthetase (OAS) enzyme is induced by IFNs
-> activated by dsRNA -> synthesizes 2'-5' oligoA -> activates RNase L -> degrades
mRNA -> no protein synthesis
-oligoA: adenosine linked together by P binding to OH on 2' of ribose instead of usual 3'
virus responds:
ex) influenza NS1: binds dsRNA, preventing OAS activation and blocking induction of
OAS by diff mechanism
interferons: IFN pathway btwn cells - Answer 1) sensors in host cell detect virus
2) sensors interact w/ IRF3, IRF7 (interferon regulatory factors) which are activated and
translocates to nucleus, activating IFN transcription (IRF3 = beta, IRF7 = alpha)
3) IFNs produced, taken up by nearby cells
(host cell dies b/c not primed, IFN beta not made fast enough to protect this cell)
4) intracellular signaling in the nearby cells, activating ISG (interferon stimulated genes)
(second cell is primed, but it may die b/c not enough OAS/PKR; third cell will inhibit
translation properly and get protected)
5) activates PKR/OAS pathways
= interferons limit viral SPREAD, not necessarily clearing the virus
interferons: influenza resistance to IFNs - Answer influenza: blocks w/ NS1
1) initial cell
-NS1 blocks, so not as much IFN beta produced = progeny virus still released
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