COMPARISON OF DEOXYOLIGONUCLEOTIDE AND TRNA(LYS-3) AS PRIMERS IN AN ENDOGENOUS HUMAN IMMUNODEFICIENCY VIRUS-1 IN-VITRO REVERSE TRANSCRIPTION TEMPLATE-SWITCHING REACTION

We developed an endogenous in vitro reverse transcription assay to stu dy the properties of priming and template switching during human immun odeficiency virus (HIV) replication. Reactions were primed with HIV re verse transcriptase (RT) and either a deoxyoligonucleotide primer (dPR ) or tRNA(Lys-3), the natural primer for reverse transcription. The RN A templates utilized were the actual HN sequences involved in the firs t template switch, namely a primer binding sequence (PBS)/ U5/R RNA do nor template and a R/U3 RNA acceptor template. Reverse transcription r eactions using the latter templates and dPR or tRNA(Lys-3) as primers yielded four major products: (-)-strong-stop DNA, a partial template-s witched DNA, full template-switched DNA, and a pseudo-PBS-primed produ ct. Use of dPR resulted in three times less template switching than wa s obtained with tRNA(Lys-3). When reactions were primed with either dP R or tRNA(Lys-3), increases in acceptor:donor template ratios resulted in augmented template switching. Increasing the concentration of RT r esulted in increased priming from the PBS but had no effect on the eff iciency of template switching. Decreasing the extent of R region overl ap resulted in a drop in efficiency of template switching. Decreases i n the R region on the donor template also caused a drop in initiation of transcription that was primed by tRNA(Lys-3) from the PBS. In contr ast, a corresponding reduction of the R region on the acceptor templat e had no effect on priming. We conclude that a transcriptional complex of tRNA(Lys-3) and RT may be associated not only with the PBS but als o with other cis RNA sequences and secondary structures in a manner es sential for efficient priming and template switching.