9: Reverse Transcription & Integration

Question 1

Which viruses require reverse transcription?

Answer 1

Gapped dsDNA: Hep B virus
ssRNA w/ DNA: Retrovirus

Question 2

Provirus Hypothesis

Answer 2

• RNA tumour caused permanent changes in cells
• Viral RNA (enzyme RNA to DNA) is integrated into host genome to become a part of it

(-)RNA viruses: RdRp in particle
(+) RNA viruses: no RdRp in particle

RT/RdDp: requires primer DNA or RNA comes from viral genome

Question 3

HIV-1 Reverse transcriptase

Answer 3

2 Active Sites
- Polymerase (palm) and RNase H:
-RNase: degrades RNA as it comes out

Question 4

Reproductive Cycle of Retrovirus

Answer 4

Need 2
1) Fusion
2) RT in the nucleocapid
3) DNA to nucleus (provirus= integration)
4) Host RNA pol transcription

virions: 2 copies of RNA, maturation by protease (cleave Gag)

proviral DNA: genomic mRNA, Gag-pol mRNA, spliced Env mRNA

Question 5

Genomic Organization of provirus

Answer 5

• After infection retroviral RNA is transcribed into DNA which is integrated into host genome (proviral DNA)

1) Genomic mRNA
2) Gag-pol mRNA codes for many proteins
3) Single spliced envelop mRNA codes for envelop proteins

tRNA from the host binds primer binding sites (PBS) on viral RNA

Question 6

How do you integrate viral dsDNA into host genome?

Answer 6

• Nucleocapsid has to allow entry of dNTPs
• Viral RNA never leaves nucleocapsid
  -dsDNA is barrier to auto integration factor (BAF), prevents integration into viral DNA
  —->dsDNA is transported to nucleus where its integrates into host genome with the help of integrate + BRD protein
• Proviral DNA directs host transcription machinery to create copies of viral mRNA
• Viral mRNA is translated to make viral proteins

Question 7

Hepatitis B Virus infectious cycle

Answer 7

• enters nucleus for repair = CCC DNA -> mRNAs (no integration)
• encapsidate mRNA + RT -> synthesis ( insufficient DNTPS = gapped)

same mRNA/envelope from the ER

Question 8

RT DNA synthesis: cytoplasmic

Answer 8

• host tRNA binds primer binding site (PBS)
• synthesize: - DNA from + RNA (RNA degraded by RNase H)
• anneal complementary sequences to 3’ end of RNA
• undegraded RNA = polypurine tract (ppi) -> primer for 2nd strand (+)
• PBS copied twice (RNA genome + tRNA) -> anneal = ds DNA with LTR (primer is removed by RN ase)
• integration of viral ds DNA (provirus)
• nucleocapsid permeabilized for dNTP entry (viral RNA never leaves)
• dsDNA leaves and BAF prevents integration (in nucleocapsid)