HIV (7-9) Flashcards
What class of virus is HIV?
HIV = Human Immunodeficiency virus
Class 6 viruses: Retroviruses
→ +ve sense ss RNA genome
→ replicate via a dsDNA intermediate - reverse transcription
Where has the heaviest burden of HIV?
Subsaharan Africa
→ 38 million people infected worldwide (more people injected and less people dying), 1.5 million new infections in 2021
→ 650,000 deaths from AIDs (15% children) in 2021
What does HIV cause?
The destruction of T cells of the immune system
→ makes the body very susceptible to opportunistic pathogens
When blood contains less than 200T cells/ul AIDS develops
What is the general life cycle of HIV?
Receptor binding
→ fuses with plasma membrane
→ deposits caspid in cytoplasm
→ uncoated
→ reverse transcription
→ new ds DNA intermediate (provirus)
→ provirus imported into nucleus
→ integrated into host cell genome
→ transcribed by host Pol II
→ viral RNAs made
→ exported out and translated
→ assembly at membrane
→ budding and maturation
What is the HIV genome comprised of?
Encodes 9 genes, 15 proteins (some encode poly proteins)
gag → structural proteins: matrix, caspid, nuclear caspid
pol → enzymes: protease, reverse transcriptase, integrase
env → envelope proteins, transmembrane
RNA ~10kb
What does a HIV particle consist of?
Diploid → 2 copies of viral RNA in each particles
Caspid → icosahedral
Nuclear caspid → coats viral RA+NA
Surrounded by matrix, which binds lipid membrane
gp120 and gp41 on the surface
What is involved in HIV receptor binding?
CD4 binding → gp120 subunits bind CD4 on T cells
→ causes conformational change in gp120
Exposure of co-receptor binding site → variable regions move out exposing highly conserved area for coreceptor binding
Co-receptor binding → binding site binds to co-receptor CCR5
How can variable regions of HIV help it to evade the immune system?
V1/2/3 - variable regions on surface of HIV envelope
→ involved in binding
→ if recognised by an antibody - v change so it doesn’t next time
What is involved in HIV fusion and entry?
gp41 of HIV has a fusion peptide - inserted into cellular membrane
→ conformational change forms hairpin - alpha helices come together
→ bring 2 membranes close enough together so they fuse
Fusion allows caspid to move into cytoplasm
T-20 fusion inhibitor blocks conformational change in gp41 → last resort treatment- nasty side effects
What is involved in HIV uncoating?
After fusion caspid moves into cytoplasm - needs to dissemble requiring chaperones
Cyclophilin A is a cellular chaperone protein that binds caspid proteins
→ incorporated into caspid structure when being made
→ enable uncoating of viral core for next step - reverse transcription
Without cyclophilin A caspid can’t uncoat - viral life cycle stops
What is reverse transcriptase?
Enzyme involved in reverse transcription of HIV
→ enters the cell in particle; incoming genome has no chance to be translated to give new enzyme
→ incoming genome not translated directly
RT protein has 2 domains:
Polymerase → nucleotides added
RNaseH → degrades RNA specifically when in a heteroduplex with a DNA strand
What are the steps of reverse transcription for HIV?
- (-) strand primed by tRNA bound at tb (tRNA binding site)
→ DNA synthesis requires a primer, for HIV this id tRNA lysine - RNase H exposes the DNA copy of R and U5 (removes the RNA complementary to the DNA)
- Base pairing between R’ and the 2nd copy of R - 1st template jump
- Continued (-) strand synthesis and template RNA degradation
→ polyP region more resistant to RNaseH activity - acts as primer to star synthesising other DNA strand - (+) strand synthesis uses (-) DNA as a template and continues until a modified a modified base in tRNA blocks extension
→ RNaseH removes polyP and tRNA - no RNA, 2 partial DNA strands - 2nd template jump → tb and tb’ at 3’ ends of (+) and (-) strands base pair
- Extension of both strands to completion
Reverse transcriptase can extend both strands → forming DNA provirus, dsDNA with extra sequence
How is HIV imported into the nucleus?
After reverse transcription more proteins bind to allows transport through nuclear pore complex
Provirus + 2 copies of integrase + matrix protein + Vpr = pre-intergration complex
Vpr → key role in nuclear transport, lacking Vpr - can’t enter nucleus
How is HIV integrated into target DNA?
LTRs bound to integrase → catalyse joining of ends to host
- Processing (integrase)
- Joining (integrase)
- Repair (host cell)
→ disrupts host cell chromosomes
→ no specific sequences, HIV integrated ay just actively transcribed areas
→ additional sequences can be added in
Is infection by proviruses cytolytic?
No
Proviruses genomes are integrated into host chromosome → infection doesn’t cause lysis
Provirus will replicate with the host genome at every round of division and propagate to every daughter cell
→ provirus can be inherited between generations if virus infects a germ cell
→ genomes of humans and other species contain large amounts of vestigial retroviral sequences
What are the long terminal repeats (LTR) of HIV genomes?
Have a role in transcription and gene expression
3 regions of sequence: U3, R, U5
5’ U3 → acts as enhancer and promoter
→ transcription starts at beginning of R region
→ lots of binding sites for cellular transcription factors - required to recruit the RNA polymerase
3’ U5 → site of poly A addition
→ transcription produces full length viral genomic RNA
What is the function of Tat protein in HIV replication?
Tat is a trans activator of transcription
→ transcription is inefficient in the absence of Tat
Tat gene is essential for HIV infection → if you delete gene virus not viable
→ in the promoter elements targeted by Tat, TAR (Tat-response) element was found immediately downstream from transcription start site
→ usually TAR function is absolutely position and orientation dependant - suggests TAR sequence functions as RNA
How does protein bind to TAR region of HIV?
TAR RNA folds into stem loop structure
→ Tat binds to bulge of stem loop
→ loop-specific factor binds above - another required factor that needs to bind to TAR
(TAR at 5’ end of RNA - transcription start site)
What is the mechanism of Tat action in HIV transcription?
In the absence of Tat:
→ the HIV TLR assembles a poorly processive RNA polymerase complex (not efficient at transcribing)
→ once clear of the promoter, RNA pol frequently drops off the template producing a truncates RNA (comprised of TAR region then falls off)
In the presence of Tat:
→ the RNA polymerase complex is converted to a fully processive mode
→ a high proportion of initiation events lead to full-length transcription
Tat converts RNA pol II to be competent
What is the role of the loop factor in HIV transcription?
Loop factor - cyclin T1 : cdk9 complex (aka Tac - Tat associated kinase)
Activated by Tat binding to TAR
→ allows phosphorylation of pol II
→ kinase activity of CDK-9 can phosphorylate c-terminus of RNA pol II - important for its activity
→ for Tat-activated elongation
How is the HIV-1 RNA genome spliced?
Un-spliced 9kb → encodes Gag, Pol
Singly spliced 4kb → encodes Vif, Vpr, Vpu, Env
Mutiply spliced 2kb → Tat, Rev, Nef
What is the role of Rev in the HIV life cycle?
Rev is a regulator of virion protein expression
In the absence of rev:
→ cytoplasmic mRNA for Gag, Pol and Env are reduced
→ mRNA for Tat, Nef are unaffected
implying Rev doesn’t have a transcriptional effect
Role in nuclear export of viral mRNAs
Why can some HIV mRNAs be transported to he nucleus in the absence of Rev?
Eukaryotic cellular mRNAs need to be processed to be exported to cytoplasm → includes splicing, introns removed
The 2kb class of viral RNA can just use host cell nuclear export pathway
→ they are fully spliced
→ 9kb/4kb get stuck in nucleus because they’re not fully spliced - require Rev
What is the rev-responsive element (RRE)
Sequence necessary for Rev to activate mRNA accumulation of an intron-containing construct
RRE confers Rev-dependent export on mRNAs that contain it and that would otherwise stay in nucleus
→ as a complex secondary structure
→ binds Rev at a high-affinity site, Rev then multimerises along the RNA
