During HIV reverse transcription, (؉) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3 end of tRNA Lys,3 hybridized to the viral primer binding site (PBS). Before (؉) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3 end of primer tRNA Lys,3 . Since the detailed mechanism of (؉) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV Lys,3 . Thus, modified nucleoside m 1 A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (؉) strand DNA transfer. We show that other factors such as the nature of the (؊) PBS of the acceptor template and the RNase H activity of RT also influence the efficacy of (؉) strand DNA transfer.The distinctive characteristic of retroviruses is the conversion of their RNA genome into double-stranded DNA, which is subsequently integrated into the cellular genome. Reverse transcription is a complex process composed of multiple steps catalyzed by the viral enzyme reverse transcriptase (RT) 1 (reviewed in Ref. 1). The general model of reverse transcription includes two obligatory DNA transfer reactions (Fig. 1A). RT initiates minus strand DNA synthesis by elongation of a specific cellular tRNA primer annealed to the primer binding site (PBS) and continues as far as the 5Ј end of the RNA genome, generating the so-called strong stop cDNA (ss-cDNA). The concomitant degradation of the RNA template by the RNase H activity of RT makes the ss-cDNA single stranded. ss-cDNA is transferred by NC protein (2, 3) to the 3Ј end of either the template RNA or the other RNA molecule present in the virus particle to complete (Ϫ) strand DNA synthesis. (ϩ) strand synthesis is primed by an RNase H-resistant sequence, the polypurine tract (PPT), and continues as far as a doublestranded RNA region corresponding to the 18-nt tRNA-viral PBS RNA complex. RT needs to unwind this tRNA-PBS RNA to reverse-transcribe the 3Ј end of primer tRNA. The stop signal for this (ϩ) strand synthesis has been proposed to be the methylated adenosine at position 58 (m 1 A-58) of primer tRNA (4, 5) but it is not the absolute termination site of RT in the case of HIV (6, 7). Termination at m 1 A-58 results in the (ϩ) strong stop DNA species carrying a complete (ϩ) PBS sequence at its 3Ј terminus. Before (ϩ) strand DNA transfer, the tRNA primer is thought to be removed by the RT-associated RNase H activity (7-11); thus, the (ϩ) PBS at the 3Ј end of (ϩ) ssDNA becomes available for hybridization to the (Ϫ) PBS synthesized at the 3Ј end of the (Ϫ) strand DNA, and synthesis of both strands resumes with strands copying each other (Fig. 1A).In this report, we analyzed the following factors that influence the efficiency of HIV-1 (ϩ) strand DNA transfer: the modified nucleosides of primer tRNA Lys,3 , the RNase H activity...