The first strand transfer reaction of HIV-1 reverse transcription is more efficient in infected cells than in cell-free natural endogenous reverse transcription reactions.
BACKGROUND
In the presence of dNTPs, intact HIV-1 virions are capable of reverse transcribing at least part of their genome, a process known as natural endogenous reverse transcription (NERT). PCR analysis of virion DNA produced by NERT revealed that the first strand transfer reaction (1stST) was inefficient in intact virions, with minus strand (-) strong stop DNA (ssDNA) copy numbers up to 200 times higher than post-1stST products measured using primers in U3 and U5. This was in marked contrast to the efficiency of 1stST observed in single-round cell infection assays, in which (-) ssDNA and U3-U5 copy numbers were indistinguishable.


OBJECTIVE
To investigate the reasons for the discrepancy in first strand transfer efficiency between intact cell-free virus and the infection process.


METHODS
Alterations of both NERT reactions and the conditions of cell infection were used to test whether uncoating and/or entry play a role in the discrepancy in first strand transfer efficiency.


CONCLUSIONS
The difference in 1stST efficiency could not be attributed simply to viral uncoating, since addition of very low concentrations of detergent to NERT reactions removed the viral envelope without disrupting the reverse transcription complex, and these conditions resulted in no improvement in 1stST efficiency. Virus pseudotyped with surface glycoproteins from either vesicular stomatitis virus or amphotrophic murine leukaemia virus also showed low levels of 1stST in low detergent NERT assays and equivalent levels of (-) ssDNA and 1stST in single-round infections of cells, demonstrating that the gp120-mediated infection process did not select for virions capable of carrying out 1stST. These data indicate that a post-entry event or factor may be involved in efficient HIV-1 reverse transcription in vivo.
Hooker
C William
CW
HIV-1 Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston, Qld 4029, Australia. b.hooker@mailbox.uq.edu.au
Harrich
David
D
D002460:Cell Line; D002474:Cell-Free System; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D003850:Deoxyribonuclease I; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic
D004279:DNA, Viral; D003850:Deoxyribonuclease I