PREFERENTIAL INCORPORATION OF NUCLEOSIDE ANALOGS AFTER TEMPLATE SWITCHING DURING HUMAN-IMMUNODEFICIENCY-VIRUS REVERSE TRANSCRIPTION

We assessed the effects of 3'-azido-3'-deoxythymidine (AZT), 2',3'-did eoxyinosine (-), and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidi ne (3TC) on reverse transcription in CD4-positive cells by isolating t runcated human immunodeficiency virus (HIV DNA fragments. Jurkat cells were treated with AZT (2 mu M), ddI (200 mu M), or 3TC (50 mu M) prio r to infection with HN. Low-molecular-weight DNA was isolated and ampl ified by PCR with primer pairs which identify different segments of HI V proviral DNA. We found that the HIV DNA fragments generated from dru g-treated, HIV-exposed Jurkat cells were truncated at a ratio of 15:1 [i.e., (-) strong-stop DNA to HIV DNA generated after the first templa te switch]. Full-length DNA was observed in the case of untreated, HIV -infected cultures. Following nucleoside analog treatment of HIV-expos ed Jurkat cells, reverse transcription was terminated only after the s ynthesis of (-) strong-stop DNA. The nucleoside analogs tested, i.e., AZT, ddI, and 3TC, preferentially chain terminated viral DNA synthesis immediately following the first template switch. The (-) strong-stop HIV DNA was present in AZT-treated and untreated cultures for at least 6 days. We also carried out cell-free reverse transcription/template- switching reactions involving tRNA(Lys3) Or a deoxyoligonucleotide as a primer, as a means of studying the selective incorporation of AZT tr iphosphate into proviral DNA. When reactions were primed with tRNA(Lys 3), We found that AZT triphosphate was preferentially incorporated aft er template snitching.