Journal of General Virology (1995), 76, 1675-1686. Printed in GreatBritain 1675 Stepwise analysis of reverse transcription in a cell-to-cell human immunodeficiency virus infection model: kinetics and implications Litsa Karageorgos, 1 Peng Li 1. and Christopher J. Burrell 1,2 1National Centre for HIV Virology Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide 5000, South Australia and 2Department of Microbiology and Immunology, University of Adelaide, North Terrace, Adelaide 5000, South Australia, Australia We have investigated the kinetics of human immuno- deficiency virus (HIV) reverse transcription in infected T cells, using a synchronized, one-step, cell-to-cell in- fection 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. Introduction One of the key early events in retrovirus replication is the reverse transcription of the single-stranded viral RNA into double-stranded DNA, which is catalysed by the virus-encoded reverse transcriptase. This process can be divided into several discrete steps: (i) synthesis of a short stretch of DNA ('the minus-strand strong-stop DNA') from the primer binding site (PBS) near the 5' end of one genomic viral RNA molecule, using a tRNA as primer; (ii) transfer of the minus-strand strong-stop DNA from the 5' end of the viral RNA to the 3' end of the same, or a second molecule of viral RNA; (iii) continued synthesis of the minus-strand DNA, accompanied by degradation of the RNA template by RNase H activity of the viral reverse transcriptase [this process leaves a polypurine tract (PPT) at specific sites in the 3' end of the viral RNA to serve as the primer for the synthesis of the plus-strand viral DNA using the minus-strand DNA as template]; * Author for correspondence.Fax + 61 8 228 7538. e-mail jmcinnes @microb.adelaide.edu.au (iv) this newly synthesized short stretch of plus-strand DNA ('plus-strand strong-stop DNA') is then trans- ferred from the 5' end to the 3' end of newly made, partially completed minus-strand DNA; (v) after the second template transfer, the remainder of the plus- strand DNA as well as the 3' end of the minus-strand DNA is synthesized, to yield a linear double-stranded DNA with all sequences in the viral RNA plus duplicated U3 and U5 sequences present in the long terminal repeats (for reviews see Varmus & Brown, 1989; Coffin, 1990; Li et al., 1993; Jones et al., 1994). Although these basic steps have been known for some time from studies of type C retroviruses [some aspects of these have recently been confirmed for the human immunodeficiency virus (HIV)] a detailed kinetic study to delineate the multiple events in this process has been lacking and the current data are highly controversial. Studies on type C retroviruses showed that the first complete linear DNA molecules could be detected approximately 4 h after infection (Varmus & Swanstrom, 1984, 1985; Varmus & Brown, 1989). For HIV, our laboratory and others using one-step virus growth conditions showed by infection with either cell-free virus 0001-3007 © 1995 SGM