HIV Reverse Transcriptase and Mutation

Question 1

HIV reverse transcription steps

Answer 1

1. HIV has 2 copies of RNA, which are reverse transcribed into dsDNA (contain long terminal repeats, LTRs, at each end) by RNA-dependent polymerase
2. Integration of cDNA into host genome by integrase enzyme
3. Genome is transcribed into RNA
4. RNA is packaged into a virus, which host cell releases

Question 2

AZT (drug) inhibits what part of the HIV cycle?

Answer 2

Reverse transcription

Question 3

Components of LTR

Answer 3

R (repeat)
U5 (unique 5)
PBS (primer binding site)

Question 4

Detailed HIV cycle steps

Answer 4

1. tRNA primer (of host origin) binds to 5’ LTR
2. R and U5 of LTR are made into DNA (“strong stop”)
3. RNase H recognizes RNA/DNA duplex and degrades RNA
4. Strong stop DNA makes 1st jump to 3’ end (R of strong stop DNA binds to R of 3’ LTR)
5. Reverse transcriptase extends the DNA
6. RNase H removes RNA
7. PPT (3’ LTR’s equivalent to PBS) acts a primer for extension of the 2nd strand
8. RNase H removes tRNA primer
9. 2nd jump: DNA is looped so PBSs of 5’ and 3’ ends can base pair
10. DNA can be extended and integrated into host genome by integrase
11. Transcription and translation of DNA
12. Budding and release of viral proteins

Question 5

Main problem of HIV

Answer 5

HIV rapidly acquires many mutations (mutates about 1000x more quickly than the human genome; about 10 of every 11 copies are completely defective)

Question 6

Why does HIV acquire so many mutations?

Answer 6

Low fidelity of reverse transcriptase
High reproduction rate
Integrase sloppiness (recombinogenic properties of provirus)
2 different copies of RNA are present to infect a cell

Question 7

Strand transfer

Answer 7

Virus contains 2 copies of its RNA genome (may not be identical)
Reverse transcriptase will jump back and forth between the RNA templates (increases frequency of recombination and provides structural diversity of virus, allowing it to evade the immune system and drugs)

Question 8

Molecular causes of strand transfer

Answer 8

Low processivity
Secondary structures (particularly hairpins, which cause reverse transcriptase to pause)
Breaks in the RNA template
RNAs are in close proximity to one another