INF2 - E. HIV LIFE CYCLE-COVERED

Question 1

what cells does HIV affect

Answer 1

• CD4+ T-cells
• lyses them during a productive infection
• leads to progressive reduction in CD4+ T-cells
• Natural killer cells
• CD8+ killer T-cells
• macrophages
• cells of nervous system
• dendritic cells
  these cells won’t be lysed, they bud out and not rupture immediately

Question 2

structure of HIV

Answer 2

• cytoplasmic tail of gp41 interacts with HIV-1 matrix protein p17
• nucleocapsid protein interacts with RNA strands

Question 3

what are the restrictions with HIV

Answer 3

• only space for a limited number of nucleotides in capsid
• limited number of genes encoded for different proteins (protease, integrate etc)
• RNA genome needed to be reverse transcribed to DNA by RT
• RT has love fidelity: if genome too long, high chance of lethal mutations for the virus and it will die out

Question 4

what are the 3 ways HIV overcomes the restrictions

Answer 4

• use of 3 reading frames
• use of poly protein precursors
• use of single capsid protein

Question 5

3 reading frames

Answer 5

• HIV genome is small: 10,000 nucleotides encoding 19 genes
• can read it at different positions which helps create and encode different proteins
• multiple reading frames allow virus to encode more and different types of proteins within same genetic material
• uses less nucleotides (ie 1 gene doesn’t = 2 protein like ours)

Question 6

poly protein precursors

Answer 6

• large precursor proteins from which smaller proteins are generated by proteolytic cleavage (protease)
• allows for more compact genome by eliminating additional genetic features eg - promoters etc for transcription factors to bind

Question 7

what are the 3 genes encoding 3 large poly proteins which are then chopped into individual subunits

Answer 7

1. Gag (for group specific antigen) - capsid and matrix
2. pol (for polymerase) - RT, integrase etc
3. env (for envelope) - gp120 and gp41 for attachment and fusion

Question 8

use of single capsid protein

Answer 8

• capsid is 1000 repeats of the p24 capsid protein (CA)
• hexameters, dimers and pentamers

Question 9

HIV tropism

Answer 9

• receptor on T-cells: CD4
• co-receptor on macrophages: CCR5 (chemokine co-receptor)
• co-receptor on T-lympohocytes: CXCR4
  *both co-receptors found on both cell types
• HIV infects macrophages (M-trophic or R5 subtypes)
• later, switches to CD4+ T-cells (T-cell tropic or X4 subtypes)
• rapid progression to AIDS associated with this switch in co-receptor

Question 10

HIV attachment

Answer 10

• gp120 binds to CD4 on host cell
• conformational changes in gp120 allows interaction with co-receptor CCR5 or CXCR4

Question 11

HIV fusion

Answer 11

• further conformation change results in gp41 fusogenic tip insertion into cell membrane
• fusion of viral and cellular membrane

Question 12

inhibitor of gp120-CD4 binding

Answer 12

• Ibalizumab: monoclonal antibody

Question 13

inhibitor of gp120-co-receptor binding

Answer 13

• maraviroc

Question 14

inhibitor of gp41-mediated membrane fusion

Answer 14

• enfuvirtide (Fuzeon)

Question 15

HIV uncoating

Answer 15

• HIV capsid doesn’t disintegrate immediately after release into cytosol
• if happens to early, infection aborted

Question 16

what are the 3 biochemical activities of retroviral RT

Answer 16

1. RNA-dependent DNA polymerase activity
   - can synthesise ssDNA from an RNA template (ss)
2. RNAseH (nuclease)
   - ribonuclease H
   - proteins which catalyse cleavage of RNA in an RNA/DNA substrate
   - degrades RNA after it’s used as a template
3. DNA-dependent DNA polymerase activity
   - can interact directly with DNA
   - copies ssDNA to dsDNA

Question 17

reverse transcriptase rules

Answer 17

1. RT can only start from double strand nucleic acid and add nucleotide in 5’ to 3’ direction
2. RT has polymerase and RNAseH activity
3. RNAseH will delete RNA in RNA/DNA duplex

Question 18

why is RT an ideal target

Answer 18

• absent in host cells

Question 19

example or a RT inhibitor

Answer 19

• Zidovudine

Question 20

NRTIs

Answer 20

• compete with naturally occurring nucleosides to prevent their incorporation into viral DNA
• prevents RT
• uncompleted ssDNA

Question 21

NNRTIs

Answer 21

• binds to RT
• prevents enzyme being able to convert viral RNA into DNA so RT process inhibited

Question 22

HIV integration

Answer 22

• integrase needed
• encoded in pol gene
• site of integration in human gene isn’t random
• inserted into regions that are actively transcribed
• ie: high G/C content, gene density

Question 23

integrase inhibitor

Answer 23

• raltegravir
• targets integrase viral DNA complex in pre-integration complex
• blocks integration of HIV so inhibits viral replication

Question 24

gag poly protein

Answer 24

• gag gene encodes structural proteins of capsid
• expressed as single-chain poly protein which has 5 cleavage sites for HIV protease
• can therefore make more capsid proteins and receptors etc
• poly protein inserted into cell membrane and cleaved in subsequent steps during maturation
• cleavage by protease results in release of matrix proteins (MA), capsid protein (CA), protein NC and p6 (initiates budding)
• sp1 and sp2 are short spacer peptide with unclear functions
• if haven’t undergone full maturation by proteolytic cleavage then viral particles aren’t infective

Question 25

example of a protease inhibitor

Answer 25

Saquinavir not cleaved by protease as they remain in the active site and block it