Retrovirus question

[Olav Hungnes]

n.panjwani at ic.ac.uk wrote:
: I have two questions about HIV that arose in a discussion I had today, and
: we reached no satisfactory conclusion. Perhaps one of you is
: pre-enlightened:

: [1] When HIV RNA is reverse transcribed, why is the polyA tail not
:     transcribed to polyT?
Minus-strand synthesis does not start at the extreme 3' end. It starts 
from a tRNA primer annealed to a primer binding site (PBS) a couple of 
hundred nt from the 5'end of the genomic RNA.
The first and last ca.100 bases (that is, last 100 bases before the polyA 
tail) of the virus RNA are identical (R regions). This means that the 
newly synthesised cDNA initiated at the PBS is complementary to the  
downstream R region also, and anneals to this during the "first jump" of 
retroviral reverse transcription. From this downstream position, 
minus-strand synthesis proceeds leftwards, probably until it has 
reached and copied the 5' end of the PBS. 
The polymerase and RNaseH activities reside on the same enzyme, and the 
RNA moiety of the RNA-DNA duplex is probably degraded shortly after it 
has been copied.
Initiation of plus strand synthesis occurs from the two polypurine tracts 
(PPTs), whose characteristics seem to make them immune to RNaseH 
degradation, so that a RNA PPT-oligo is left on the DNA minus strand and 
act as plus-strand primers (this is what I used to work on).
The second and final "jump" occurs upon hybridization between the 
resulting terminal PBS sequences, allowing  further elongation of the 
plus and minus strands to form a full-length double-stranded proviral DNA.
(I really should have made a drawing).

: [2] How many copies of provirus can exist in a cell, i.e. how many HIV cDNAs
:     can integrate? Does multiplicity have any known effect on pathogenesis?

A cell infected in cell culture can harbor as many as 80 proviruses. This 
happens multiple (secondary) infection events, as a anti-CD4 MAb which 
blocks infection will also block this provirus accumulation when added to 
the cells immediately after the initial infection. 
What happens in vivo, and has it any significance in pathogenesis? I 
don't know.

Olav

: Thank you for your time,

: Naveed Panjwani
: Dept of Clinical Sciences
: London School of Hygiene and Tropical Medicine
: University of London, UK.

: E-mail:  n.panjwani at ic.ac.uk


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Olav Hungnes                 olav.hungnes at embnet.uio.no
National Institute               Phone  (+47)22042200
of Public Health                 FAX    (+47)22353605
Oslo, NORWAY
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