L7 - HIV (3) Flashcards
What are intracellular inhibitors of HIV replication?
TRIM5a APOBEC3 Tetherin SAMHD1 SERINC3/5 NONO
What are the 2 alleles/variants of MLV?
N-tropic = N-MLV B-tropic = B-MLV
infected inbred mice
What was the factor that was tested for in MLV?
Fv1 (N or B)
What does Fv1 do?
mediated post-entry block to viral replication
blocks from going into the nucleus
What is Fv1?
60% homology to MLV capsid protein
single aa change in CA - alters tropism form N to B
What happens when HIV-1 enter a Rhesus monkey cell?
blocked BEFORE reverse transcription
cannot be Fv1 as before the reverse transcription
What was the name of the experiment carried to find out why HIV-1 did not replicate in Rhesus monkeys?
cell fusion experiment
HIV could NOT replicate in the human/monkey fusions
What was the method for identifying the HIV-1 restriction factor TRIM-5a?
cDNA library
transfection
infect with HIV-GFP and select the GFP-neg ones
expand the GFP-neg ones
PCR with specific primers
clone and sequence
What is the HIV-1 restriction factor?
TRIM5a
What is TRIM5a?
- RING and B-box = ubiquitination, rapid proteasomal degradation
- Coiled coils - dimerise
- SPRY domain - can bind to HIV capsid antigen
What is the structure of the HIV-1 capsid?
12 pentamers
215 hexamers
What is the mutation in the Capsid antigen that prevents the TRIM5a block?
Mutate Arg110 to Glu
What is the mechanism of TRIM5a restriction?
2-stage mechanism
dimerised coiled coil binds to the capsid through SPRY domain - forms net, blocks infection
ubiquitination stops reverse transcription and causes capsid disruption
How does TRIM5a form a net around the capsid?
SPRY domains interacts with the capsid
What is the 2-stage mechanism of TRIM5a restriction?
infection is blocked due to binding
reverse transcription is blocked due to ubiquitination
How does human TRIM5a NOT block HIV-1?
mutation in SPRY domain preventing the interacting with HIV-1 CA
How did HIV-1 cross over from SIVcpz concerning TRIM5a?
SIVcpz is NOT blocked by human TRIM5a
What is Vif protein?
cytoplasmic
192aa
What is the function of Vif protein?
COUNTERACTS APOBEC3G
affects virus maturation (enhances infectivity)
Binds to virion RNA in cytoplasm - incorporates into virions
How was reverse genetics analysis of vif carried out?
introduce stop codon
disocvered phenotype of Vif mutant = CELL-TYPE DEPENDENT
NON-INFECTIOUS IN NON-PERMISSIVE CELLS
What is the cellular antiviral factor that Vif counteracts?
APOBEC3G
What is APOBEC3G?
ONLY expressed in NON-PERMISSIVE CELLS
cytosine deaminase
deaminates cytosine in uracil
What happens to APOBEC3G in the absence of Vif?
APOBEC3G incorporated into virus particles
What is a non-permissive cell?
A cell that does NOT allow the multiplication within it of a virus
What does APOBEC3G actually do?
deaminates a SINGLE cytosine in APOB mRNA
replaces the NH group with oxygen
convert the CYTOSINE to a URACIL (not normally present in DNA)
HYPERMUTATIONS as you will get base A incorporated into chain instead of G
MISREPLICATION AND DEGRADATION BY ENZYME UNG2 (Ffor when uracil is incorporated into DNA)
How does Vif block the action of APOBEC3G?
Ubiquitin ligase complex
targets it for proteasomal degradation
What is Vpu?
81aa
membrane protein
What is the function of Vpu?
binds to CD4
induces degradation by ubiquitination
Env escapes being trapped in ER
enhances virion release - would get stuck on cell surfaces otherwise
Is Vpu present in HIV-1 and HIV-2?
NO - only HIV-1
How is the phenotype of HIV-1 Vpu mutant cell-type dependent?
virus released in permissive cells
virus retained on cell surface in non-permissive cells
What is Tetherin?
Cell surface protein
blocks enveloped virus release
How can tetherin expression be stimulated?
IFN treatment
How does Vpu interact with tetherin?
ubiquitination degradation
What does the deletion in the cytoplasmic tail oh human tetherin mean?
cannot interact with Nef protein like SIVcpz and SIVgor can
Why is M group more successful than HIV-1 O, N & P?
HIV-1 M Vpu can interact with tetherin in diff. way as it Nef cannot interact with tetherin and pull it away from membrane
What is SAMHD1?
^ expressions in myeloid cells
early stage block
only functions in quiescent cells
target for HIV-2 Vpx
What autoimmune condition results from mutated SAMHD1?
Aicardi-Goutiere Syndrome
dysregulation of IFN system
how does SAMHD1 work?
dGTP activates
cleaves triphosphate from deoxyribonuceloside triphosphates
removes triphosphate from pool of dNTPs
inhibits REVERSE TRANSCRIPTION
What are SERINC 3/5?
Serine incorporators
transmembrane
serine into lipids
What protein prevents incorporation of SERINC3/5 into virus?
Nef
What is NONO?
Nuclear CAPSID sensor
links to viral DNA sensor - cGAS
WORKS IN THE NUCLEUS
INDUCES INNATE IMMUNE RESPONSE
