Stepwise analysis of reverse transcription in a cell-to-cell human immunodeficiency virus infection model: Kinetics and implications

L. Karageorgos, P. Li, C. J. Burrell

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

We have investigated the kinetics of human immunodeficiency virus (HIV) reverse transcription in infected T cells, using a synchronized, one-step, cell-to-cell infection model and quantitative PCR assays for the different DNA intermediate structures that are found sequentially during reverse transcription. Different efficiencies that might arise from the use of different primers and other PCR conditions were normalized by conversion of each PCR product signal to copy numbers by comparing with standards. After an initial lag period, the minus-strand strong-stop viral DNA was detected first. This was followed by the post-transfer newly extended minus-strand viral DNA and then by the plus-strand strong-stop DNA and fully extended minus-strand DNA. Kinetic data indicated that, once reverse transcription was initiated, the HIV reverse transcriptase synthesized minus-strand DNA at a rate of 150-180 bases/min, and that the first template transfer and the initiation of the plus-strand DNA synthesis imposed specific time delays. In contrast, minus-strand viral DNA synthesized after the second template transfer appeared at a time point very close to the time of the appearance of the last piece of DNA synthesized just before the second template switch, suggesting that the second switch occurred very rapidly. Taken together, our results define more accurately than was previously possible the rates of several of the steps in HIV reverse transcription in infected T cell lines and indicate different mechanisms for the two distinct template switches during retrovirus reverse transcription.

LanguageEnglish
Pages1675-1686
Number of pages12
JournalJournal of General Virology
Volume76
Issue number7
DOIs
Publication statusPublished - 1995
Externally publishedYes

ASJC Scopus subject areas

  • Virology

Cite this

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abstract = "We have investigated the kinetics of human immunodeficiency virus (HIV) reverse transcription in infected T cells, using a synchronized, one-step, cell-to-cell infection model and quantitative PCR assays for the different DNA intermediate structures that are found sequentially during reverse transcription. Different efficiencies that might arise from the use of different primers and other PCR conditions were normalized by conversion of each PCR product signal to copy numbers by comparing with standards. After an initial lag period, the minus-strand strong-stop viral DNA was detected first. This was followed by the post-transfer newly extended minus-strand viral DNA and then by the plus-strand strong-stop DNA and fully extended minus-strand DNA. Kinetic data indicated that, once reverse transcription was initiated, the HIV reverse transcriptase synthesized minus-strand DNA at a rate of 150-180 bases/min, and that the first template transfer and the initiation of the plus-strand DNA synthesis imposed specific time delays. In contrast, minus-strand viral DNA synthesized after the second template transfer appeared at a time point very close to the time of the appearance of the last piece of DNA synthesized just before the second template switch, suggesting that the second switch occurred very rapidly. Taken together, our results define more accurately than was previously possible the rates of several of the steps in HIV reverse transcription in infected T cell lines and indicate different mechanisms for the two distinct template switches during retrovirus reverse transcription.",
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Stepwise analysis of reverse transcription in a cell-to-cell human immunodeficiency virus infection model : Kinetics and implications. / Karageorgos, L.; Li, P.; Burrell, C. J.

In: Journal of General Virology, Vol. 76, No. 7, 1995, p. 1675-1686.

Research output: Contribution to journalArticle

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