HIV (7-9)

Question 1

What class of virus is HIV?

Answer 1

HIV = Human Immunodeficiency virus

Class 6 viruses: Retroviruses
→ +ve sense ss RNA genome
→ replicate via a dsDNA intermediate - reverse transcription

Question 2

Where has the heaviest burden of HIV?

Answer 2

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

Question 3

What does HIV cause?

Answer 3

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

Question 4

What is the general life cycle of HIV?

Answer 4

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

Question 5

What is the HIV genome comprised of?

Answer 5

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

Question 6

What does a HIV particle consist of?

Answer 6

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

Question 7

What is involved in HIV receptor binding?

Answer 7

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

Question 8

How can variable regions of HIV help it to evade the immune system?

Answer 8

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

Question 9

What is involved in HIV fusion and entry?

Answer 9

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

Question 10

What is involved in HIV uncoating?

Answer 10

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

Question 11

What is reverse transcriptase?

Answer 11

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

Question 12

What are the steps of reverse transcription for HIV?

Answer 12

1. (-) strand primed by tRNA bound at tb (tRNA binding site)
   → DNA synthesis requires a primer, for HIV this id tRNA lysine
2. RNase H exposes the DNA copy of R and U5 (removes the RNA complementary to the DNA)
3. Base pairing between R’ and the 2nd copy of R - 1st template jump
4. Continued (-) strand synthesis and template RNA degradation
   → polyP region more resistant to RNaseH activity - acts as primer to star synthesising other DNA strand
5. (+) 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
6. 2nd template jump → tb and tb’ at 3’ ends of (+) and (-) strands base pair
7. Extension of both strands to completion

Reverse transcriptase can extend both strands → forming DNA provirus, dsDNA with extra sequence

Question 13

How is HIV imported into the nucleus?

Answer 13

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

Question 14

How is HIV integrated into target DNA?

Answer 14

LTRs bound to integrase → catalyse joining of ends to host

1. Processing (integrase)
2. Joining (integrase)
3. Repair (host cell)

→ disrupts host cell chromosomes
→ no specific sequences, HIV integrated ay just actively transcribed areas
→ additional sequences can be added in

Question 15

Is infection by proviruses cytolytic?

Answer 15

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

Question 16

What are the long terminal repeats (LTR) of HIV genomes?

Answer 16

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

Question 17

What is the function of Tat protein in HIV replication?

Answer 17

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

Question 18

How does protein bind to TAR region of HIV?

Answer 18

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)

Question 19

What is the mechanism of Tat action in HIV transcription?

Answer 19

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

Question 20

What is the role of the loop factor in HIV transcription?

Answer 20

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

Question 21

How is the HIV-1 RNA genome spliced?

Answer 21

Un-spliced 9kb → encodes Gag, Pol
Singly spliced 4kb → encodes Vif, Vpr, Vpu, Env
Mutiply spliced 2kb → Tat, Rev, Nef

Question 22

What is the role of Rev in the HIV life cycle?

Answer 22

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

Question 23

Why can some HIV mRNAs be transported to he nucleus in the absence of Rev?

Answer 23

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

Question 24

What is the rev-responsive element (RRE)

Answer 24

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

Question 25

How does Rev-mediated HIV RNA export occur?

Answer 25

Rev binds viral RNA (contains envelope coding region - not fully spliced)
→ binds the exporting Crm 1 and exits dependant on RAN

2kb able to exit in absence of Rev → producing Rev, Tat and Nef which go back into nucleus
→ Tat activates more transcription
→ Rev binds RRE
9kb and 4kb class can be exported in presence of Rev

HIV has 2 phases of gene expression:
Early → Rev, Tat, Nef
Late (Rev dependant) → Gag, Pol, Env, Vif, Vpu, Vpr

Question 26

How are Gag and Pol translated from the HIV RNA genome?

Answer 26

Gag and Pol are translated from the full length 9kb HIV RNA
→ the two proteins are in different overlapping reading frames
→ ribosomal frame shifting

Ribosomes bind at 5’ end, start translating Gag
→ 9/10 ribosomes translate entire protein
→ 1/10 ribosomes when they reach frameshift signal pause, shift back one nucleotide
(frameshift signal near end of Gag open reading frame)
→ continue translating in Pol reading frame
→ produces Gag-Pol fusion poly protein - 3/4 proteins of Gag + 3 enzymes of Pol

Question 27

How has the translation of HIV protein frameshift efficiency evolved?

Answer 27

Viruses evolve frameshift efficiency dependant on its needs
→ to provide proteins in the relative ratios they need

90% of ribosomes produce Gag, 10% of ribosomes produce Gag-Pol fusion protein
→ Gag encodes structural proteins so 10x more needed compared to the enzymes encoded by Pol

Question 28

How does the frame shifting occur in HIV RNA?

Answer 28

A stem-loop structure downstream of slippery sequence in the HIV RNA, blocks mRNA entrance tunnel in ribosome
→ causes the ribosome to pause during translation while A and P sites over a UUUUUUA slippery sequence
→ 10% of the time the ribosome slips back 1 nucleotide then continues to translate Gag-Pol fusion protein

Question 29

How is the HIV Env protein produced?

Answer 29

Env is a transmembrane glycoprotein
→ its co-translationally inserted into ER membrane
→ gp41 region of Env traverses the membrane, gp120 region of Env in the lumen of the ER
→ gp120 heavily glycosylated in the ER - enables some of the immune evasion
→ Env follows the route of membrane and secretory proteins in vesicles via the Golgi to the cell membrane
→ Env clusters in regions of the membrane that are high in cholesterol, called lipid rafts

Question 30

What does HIV particle assembly require?

Answer 30

→ 2 copies of full length HIV RNA
→ ~2000 molecules of Gag protein
→ lipid envelope contains Env glycoproteins

Question 31

How does HIV ensure only full length RNA packaged into viral particles?

Answer 31

Full length HIV RNAs, but not spliced RNAs, contain a packaging signal (Psi)
→ located downstream of major splice site - sequence removed on spliced RNAs

Gag protein binds to Psi, then multimerises along the RNA

HIV RNAs also contain a dimerisation signal → palindromic region that allows interactions between 2 viral RNAs
→ end up with dimer of RNA molecules coated in Gag

Question 32

What is involved in HIV assembly, budding and maturation?

Answer 32

The Gag-RNA complex forms in the cytoplasm
→ transported to a region of the membrane containing Env proteins to form an immature virus
→ hijacks vesicle trafficking - required ESCRT complexes
→ enables complex to be exported Bothe the membrane

Protease self-cleaves from Gag-Pol - catalyses maturation
→ then able to cleave all the other Gag-Pol proteins into their constituent parts
→ caspid protein rearranged to get mature particle (infectious)
→ matrix protein remains bound to the membrane
→ caspid proteins form icosahedral structure surrounding viral genome - bound to nucleocapsid

Question 33

What are the roles of the HIV accessory proteins?

Answer 33

Involved in counteracting host antiviral and immune responses

Vif → inhibits host cell APOBEC3G - an antiviral enzyme that causes hypermutation of the HIV genome
Vpu → inhibits host cell tetherin - a cell surface molecule preventing the release of viral particles
Nef → causes the down regulation of MHC class I and CD4 from the cell surface - prevents cell signalling to immune system, can’t present peptides

Question 34

What is AIDS?

Answer 34

Acquired immune deficiency syndrome

Current definition:
→ the presence of 1 of 25 conditions indicative of severe immunosuppression
or
→ HIV infection with a CD4+ T cell count of <200 cells / ul of blood

First identified in 1981 → Pneumocystis pneumonia and Kaposi’s sarcoma (rare diseases linked to immunosuppression) unusual to find in clusters
→ clustered in sexually interactive groups and needle-sharing drug users - suggestive of infectious disease passed via bodily fluids

Question 35

What was the first human retrovirus discovered?

Answer 35

Human T-cell lymphotropic virus (HTLV-I) in late 1970s
→ HTLV-II discovered in 1982

Question 36

When was the AIDS virus isolated?

Answer 36

May 1983 - Montagnier and colleagues
→ isolation of a T-lymphotropic retrovirus from a patient at risk for AIDs
→ called it lymphadenopathy-assocaited virus (LAV)

May 1984 - Gallo and colleagues
→ identified a retrovirus from blood of AIDS patients
→ DNA showed homology to HTLV-I
→ called it HTLV-III
(identified same virus)

1985
→ complete genomic sequence published
→ found to be not closely related to HTLV-I
→ virus renamed human immunodeficiency virus (HIV)

1986 - Clavel and Montagnier
→ isolate HIV-2 from patients in West Africa

Question 37

What are the origins of HIV?

Answer 37

Likely to be from zoonotic transmission (trans-species) - simian to human

HIV-1 → genetic homology to a virus isolated from captive chimpanzees in 1989 - SIVcpz

HIV-2 → genetic homology to a virus isolated from captive macaque in 1985 - SIVmac
→ macaque not natural host - caught from sooty mangabey monkeys

Question 38

Where is HIV-2 mainly found?

Answer 38

HIV-2 mainly limited to West Africa
→ sooty mangabey habitats found in WA
→ low world-wide incidence
→ higher in countries with colonial ties to WA (France, Portugal)

Question 39

What species of chimpanzee did HIV-1 originate from?

Answer 39

P. troglodytes troglodytes - SIVcpzPtt → HIV-1

1000 wild chimpanzees (with 4 distinct subspecies with non-overlapping habitats) tested 1989-1999
→ most of them were P. t. verus and -ve for SIVcpz
→ the two that were found positive were P. t. troglodytes

P. troglodytes schweinfurthii - SIVcpzPts
→ not transmitted to humans - dead end host

Question 40

What four groups can HIV-1 be divided into?

Answer 40

Group M (main) → 98% of HIV-1 isolates; found worldwide
Group O (outlier) → 1-2% of HIV-1 isolates; limited to Cameroon and Gabon
Group N (new) → 6 individuals in Cameroon
Group P (2009) → 2 individuals in Cameroon
→ N and P not thought to be transmittable between humans
→ divided based on their sequence diversity

Question 41

Where did HIV-1 groups M and N arise from?

Answer 41

Different locations - separate transmission events from chimps to humans
→ N more central Cameroon
→ M more southeast Cameroon

Question 42

Where did HIV-1 groups O and P arise from?

Answer 42

Gorillas in western and eastern lowland Africa

SIVcpzPtt → SIVgor → HIV-1 group O / HIV-1 group P (separate transmission events)

Question 43

Where did SIVcpz originate from?

Answer 43

SIVcpz is a recombinant virus
→ red capped managabey (SIVrcm)
→ greater spot-nosed monkey (SIVgsn)

Properties of the recombinant virus made it efficient in chimps
→ chimps closer related to humans - advantaged for transmitting to humans

Question 44

Is HIV a zoonotic virus?

Answer 44

Zoonotic virus = an animal virus that is transmissible to man under natural conditions
Humans have been exposed to SIV-infected monkeys for thousands of years; HIV has only emerged in the lat 100 years
→ only 12 cross-species transmission events
→ suggests genetic differences between host species are barriers to infection and viral mutations required to infect new hosts

The error-prone nature of HIV reverse transcriptase allows rapid viral evolution
→ RNA dependant DNA polymerase - no proof-reading - enables adaptation
→ HIV diploid genome - reverse transcriptase can jump template - recombination

Question 45

How must a virus evolve to infect and replicate in a new host?

Answer 45

A virus must evolve to use dependency factors in the new host and evade the restriction factors

Dependancy factors → host cell proteins viruses rely on for replication - small differences between species may prevent replication
Restriction factors → mammalian cells have antiviral defences in addition to the innate immune response - host specific antiviral proteins

Question 46

What types of AIDS are asymptomatic and pathogenic in host?

Answer 46

SIV is non-pathogenic in its natural hosts (African monkeys) → co-evolution for at least 100,000 years
→ asymptomatic in host: SIVsmm, SIVrcm, SIVgsn, SIVagm

SIV/HIV causes AIDS in chimps and humans
→ pathogenic in host: HIV-1, SIVcpz, HIV-2, SIVmac

Question 47

Why does HIV-infection in humans progress to AIDS without treatment but SIV-infected monkeys are asymptomatic?

Answer 47

There is a high level of viral replication in both however

HIV in humans → T cell depletion, so opportunistic diseases can invade
→ high level of innate immune activation - inflammatory response

SIV in monkeys → T cell population persevered
→ low level innate response
→ although there is a high level of viral replication - monkeys don’t become susceptible to opportunistic diseases

Question 48

How does HIV stimulate the innate immune response in humans?

Answer 48

Through TLR7 and TLR9 in plasmacytoid dendritic cells
→ TLR7+9 signal through IRF-7 to produce IFNalpha
→ continued pDC activation and IFN production are likely to lead to the accelerated destruction and impaired regeneration of T cells

Question 49

How does HIV stimulate the immune response in monkeys?

Answer 49

Much lower levels of IFNalpha are produces in response to TLR7+9 signalling
→ monkey IRF-7 has several different aa in its activation domain compared to humans - thus less active in stimulating transcription of IFNalpha - preservation of T cells
(it is not beneficial for virus to kill host - evolution of IRF-7 in response to thousands of years of co-existence with SIV?)