STEPWISE ANALYSIS OF REVERSE TRANSCRIPTION IN A CELL-TO-CELL HUMAN-IMMUNODEFICIENCY-VIRUS INFECTION MODEL - KINETICS AND IMPLICATIONS

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 th e different DNA intermediate structures that are found sequentially du ring reverse transcription. Different efficiencies that might arise fr om the use of different primers and other PCR conditions were normaliz ed by conversion of each PCR product signal to copy numbers by compari ng with standards. After an initial lag period, the minus-strand stron g-stop viral DNA was detected first. This was followed by the post-tra nsfer newly extended minus-strand viral DNA and then by the plus-stran d strong-stop DNA and fully extended minus-strand DNA. Kinetic data in dicated that, once reverse transcription was initiated, the HIV revers e transcriptase synthesized minus-strand DNA at a rate of 150-180 base s/min, and that the first template transfer and the initiation of the plus-strand DNA synthesis imposed specific time delays. In contrast, m inus-strand viral DNA synthesized after the second template transfer a ppeared at a time point very close to the time of the appearance of th e last piece of DNA synthesized just before the second template switch , suggesting that the second switch occurred very rapidly. Taken toget her, our results define more accurately than was previously possible t he rates of several of the steps in HIV reverse transcription in infec ted T cell lines and indicate different mechanisms for the two distinc t template switches during retrovirus reverse transcription.