Virus Assembly & Release 2 Flashcards
What type of virus is HIV?
Retrovirus
How big is the RNA genome of HIV?
9.5 +ve RNA genome
Where does HIV assembly take place?
plasma membrane
intracellular vesicles
How are HIV virions released?
budding
non-cytopathic
What are components of the HIV virion?
Gag Pol Env
- gp41, gp120
- 2 x ssRNA(+)
- p24, p7, p17, p6
- accessory proteins
How do retroviruses (HIV) express their structural proteins?
Gag Pol Env
- 5’ LTR drives transcription
- ribosome falls off at end of pol
- Splice donor/acceptor
- GagPol is removed leaving shortened RNA encoding only Env
How does frameshift occur to make Gag-Pol?
- change frame at end of Gag
- change frame into ORF which codes for Pol protein
- bypass stop codon to produce fusion protein
Why do you only want a small amount (5%) of Gag-Pol fusion protein
- pol makes enzymes, do not need much
- gag is a structural protein
What are the 4 proteins made by Gag
MA matrix antigen
CA capsid antigen
NC nucelocapsid
p6
What are the 3 proteins made by Gag-pol?
Protease
Reverse transcriptase
Integrase
How is Gag targeted to membranes?
myristate does NOT interact with aa sequences
myristate inserts into bilayer
stabilised by electrostatic interaction
How is Env expressed?
- Env protein to membrane
- ribosome binds to SRP
- signal peptide cleaved off by signal peptidase
- cleavage of gp160 to gp41 and gp120 by furin
- disulfide bond & trimerise
How is genomic RNA incorporated into the HIV virion?
- packaging signal after LTR
- 4 stem loops
- bound by NC (p7) of p55Gag
- motif Cys-Cys-His-Cys (similar to zinc-finger)
How do immature HIV capsids assemble?
- 1500 p55 molecules assemble at membrane
- can occur in cell-free system
- form in insect & human cells but NOT yeast or murine (cellular factors important)
- ATP
How does cellular trafficking occur via multi-vesicular bodies?
- clathrin coated pit
- early endosome
- MVB
- exosomes - contain viral mRNA and glycoproteins on surfaces
How does the glycoprotein move to outside surface in the MVB?
- trimer interacts with clathrin due to targeting sequence in gp41
- when buds through, becomes on outside when membrane is pushed through to make the vesicles
What is the ESCRT?
- 30-40 cellular proteins
- get material into MVBs
- HIV can interact with components
How does HIV Gag recruit the ESCRT complex?
- p6 - PTAP late domain
- interacts with ESCRT complex
- Gag accumulates with ESCRT complex on cytoplasmic face of MVB
- Gag buds into MVB
How does HIV release occur via MVBs?
- MVB fuse with membrane
- non cytopathic
- normal cellular mechanism
What other way apart from MVBS can HIV be released?
- budding from plasma membrane
Gag recruits ESCRT to plasma membrane - may involve lipid rafts
How does cellular trafficking occur via multi-vesicular bodies?
- clathrin coated pit
- early endosome
- MVB
- exosomes - contain viral mRNA and glycoproteins on surfaces
How does the glycoprotein move to outside surface in the MVB?
- trimer interacts with clathrin due to targeting sequence in gp41
- when buds through, becomes on outside when membrane is pushed through to make the vesicles
What is the ESCRT?
- 30-40 cellular proteins
- get material into MVBs
- HIV can interact with components
How does HIV Gag recruit the ESCRT complex?
- p6 - PTAP late domain
- interacts with ESCRT complex
- Gag accumulates with ESCRT complex on cytoplasmic face of MVB
- Gag buds into MVB
How does HIV release occur via MVBs?
- MVB fuse with membrane
- non cytopathic
- normal cellular mechanism
What other way apart from MVBS can HIV be released?
- budding from plasma membrane
What are the accessory proteins incorporated into HIV-1 virions?
- Vpr - interacts with p6 (infectivity)
- Vif - binds RNA (infectivity)
- Nef - myristolated (infectivity)
What cellular proteins are incorporated into HIV particles?
- CyPA - peptidyl-prolyl isomerase
- target for immunosuppressive drug cyclosporin A
- incorporates MHC Class I
What are the similarities of HIV and poliovirus assembly?
- myristolation of structural proteins (VP4 vs p17MA)
- proteolytic cleavage of polyprotein
- membrane structures for assembly
