HIV Part 2 Wk 3

Question 1

After fusion, capsid trafficking to the nucleus and virus genome replication begin

Question 2

HIV replication cycle - genome replication is via a dsDNA intermediate

Answer 2

tRNA (in capsid) anneals to primer binding site (PBS) close to 5’end of genome
Reverse Transcriptase (DNA polymerase) synthesises a short (-) DNA strand from 3’ end (-OH) of tRNA primer = complementary to R & U5 sequences
Copied region of RNA genome degraded by virus ribonuclease (HIV=part of RT)
Newly synthesised (-)DNA strand re-anneals at 3’ R sequence

					= TEMPLATE SWITCHING

RT continues to extend (-)DNA strand = all genome sequences copied, to PBS region

Question 3

3’ end of RNA genome (R, U3) degraded by virus ribonuclease

RT starts to synthesis 2nd strand

= (+)DNA, synthesised from 3’-OH at end of PPT sequence (polypurine tract)

Answer 3

All remaining RNA genome sequences degraded by virus ribonuclease

Newly synthesised (+)DNA strand re-anneals at 5’ PBS sequence

					= TEMPLATE SWITCHING

RESULT is duplication of sequences = (U3-R-U5) at both ends

Question 4

RT final steps synthesises both DNA strands
Both stands act as primer for their own extension
and as template for complementary strand synthesis

Answer 4

Compare fully copied, dsDNA genome intermediate with original RNA genome

dsDNA copy has duplicated ends = Long Terminal Repeats (LTRs)

Question 5

Antiviral drug target – reverse transcriptase

Answer 5

Azidothymidine - (Zidovudine / Retrovir)
Lamivudine- (3TC)
Emtricitabine (FTA/Emtriva)
Tenofovir disoproxil - (viread)
Nucleotide analogue RT inhibitor NtRTI

Question 6

NRTI and NtRTi mechanism of action – DNA chain terminators

Question 7

Antiviral drug target – non-nucleoside reverse transcriptase inhibitors NNRTI

Answer 7

nevirapine (viramune)
rilpivirine (Edurant)
NNTRIs dont required Phosphorylation step
Bind RT at discrete site
Inhibits RT polymerase enzyme activity

Question 8

Chain terminators against Reverse Transcriptase have prophylactic activity

Tenofovir disoproxil

Answer 8

Nucleotide analogue (NtRTI)

Question 9

Chain terminators against Reverse Transcriptase have prophylactic activity
Emtricitabine

Answer 9

Nucleoside analogue (NRTI)

Question 10

2012 meta-analysis of clinical trials
Reduce risk of acquiring HIV in high-risk individuals

Answer 10

Tenofovir = 6% reduction in risk
Tenofovir + Emtricitabine = 51% reduction in risk

Tenofovir + Emtricitabine = Truvada - used for therapeutic treatment (HIV+)
and Pre-exposure prophylaxis (PrEP; HIV-)

Question 11

dsDNA intermediate integrates into cell chromosome

Answer 11

Virus genome intermediate dsDNA - held within a partially opened capsid
Traffics to nucleus bound to
-polymerase enzyme complex, integrase enzyme
-molecules of the virus matrix, Vpr + Nef proteins
=Pre-integration complex (PIC)
Capsid deforms to pass through nuclear pores

Question 12

dsDNA intermediate integrates into cell chromosome

Answer 12

Integrase - degrades 3’ ends of virus DNA to expose reactive 3’-OH

 - cuts cellular DNA at target sites (predominantly euchromatin regions)

5’ overhangs of the DNAs anneal and are ligated Cell & virus enzymes ligate DNAs
integrated DNA genome is referred to as a provirus

Question 13

Antiviral drug target – integrase

Answer 13

Raltegravir (Isentriss)
Bictegravir (BIC)
Dolutegravir (Tivicay)
Elvitegravir (EVG)
Integrase inhibitors target the catalytic site of HIV integrase to inhibit the viral integration of proviral DNA into host genomes

Question 14

HIV Provirus transcription – gag and gag-pol polyproteins
Infected cell transcribes full length RNAs from integrated provirus (dsDNA)

Answer 14

• enzyme = cellular RNA polymerase II
  
  • promoter / enhancer sequences are in the 5’ LTR (U3 sequences)
  • polyadenylation (terminator) signal is in the 3’ UTR (U5 sequences)
    Full-length messenger RNA transcripts with = (+)ssRNA with 5’ cap, 3’ polyA tail

A. Most act as normal mRNAs = bind ribosomes for translation of gag and pol proteins

B. Some traffic to plasma membrane, as new virus genomes for virion assembly

Question 15

HIV Provirus transcription – gag and gag-pol polyproteins

Question 16

Provirus transcription – env polyprotein and other proteins

Answer 16

Infected cell transcribes rest of virus genes as a set of alternately spliced sub-genomic mRNAs
Each mRNA is translated by ribosomes through normal mechanisms
Resulting ENV polyprotein is cleaved by virus protease enzyme

Question 17

Translation of gag & gag-pol polyproteins

Question 18

Polyproteins that contain several functional enzymes

Answer 18

GAG polyprotein comprises the core structural proteins

POL polyprotein comprises the replicative enzymes

INCLUDING the protease enzyme that cleaves all the polyproteins

ENV polyprotein comprises the envelope glycoproteins

Question 19

Antiviral drug target – protease inhibitors

Answer 19

Saquinavir (Invirase)
Darunavir (Prezista)