- RING and B-box = ubiquitination, rapid proteasomal degradation - Coiled coils - dimerise - SPRY domain - can bind to HIV capsid antigen

L7 - HIV (3) Flashcards by Micro Bio

What are intracellular inhibitors of HIV replication?

TRIM5a
APOBEC3
Tetherin
SAMHD1
SERINC3/5
NONO

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What are the 2 alleles/variants of MLV?

N-tropic = N-MLV
B-tropic = B-MLV

infected inbred mice

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What was the factor that was tested for in MLV?

Fv1 (N or B)

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What does Fv1 do?

mediated post-entry block to viral replication

blocks from going into the nucleus

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What is Fv1?

60% homology to MLV capsid protein

single aa change in CA - alters tropism form N to B

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What happens when HIV-1 enter a Rhesus monkey cell?

blocked BEFORE reverse transcription

cannot be Fv1 as before the reverse transcription

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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

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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

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What is the HIV-1 restriction factor?

TRIM5a

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What is TRIM5a?

RING and B-box = ubiquitination, rapid proteasomal degradation
Coiled coils - dimerise
SPRY domain - can bind to HIV capsid antigen

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What is the structure of the HIV-1 capsid?

12 pentamers

215 hexamers

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What is the mutation in the Capsid antigen that prevents the TRIM5a block?

Mutate Arg110 to Glu

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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

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How does TRIM5a form a net around the capsid?

SPRY domains interacts with the capsid

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What is the 2-stage mechanism of TRIM5a restriction?

infection is blocked due to binding

reverse transcription is blocked due to ubiquitination

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How does human TRIM5a NOT block HIV-1?

mutation in SPRY domain preventing the interacting with HIV-1 CA

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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