Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription

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 5' RNA, natural modified tRNA(Lys,3), synthetic unmodified tRNALys,3 Or oligonucleotides (RNA or DNA) complementary to the PBS, as well as the viral proteins RT and nucleocapsid protein (NCp7), Prior to (+) strand DNA transfer, RT stalls at the double-stranded tRNA-PBS RNA complex and is able to reverse-transcribe modified nucleosides of natural tRNA(Lys,3). Modified nucleoside m(1)A-58 of natural tRNA(Lys,3) is only partially effective as a stop signal, as RT can transcribe as far as the hyper-modified adenosine (ms(2)t(6)A-37) in the anticodon loop. m(1)A-58 is almost always transcribed into A, whereas other modified nucleosides are transcribed correctly, except for m(7)G-46, which is sometimes transcribed into T. In contrast, synthetic tRNA(Lys,3), an RNA PBS primer, and a DNA PBS primer are completely reverse-transcribed, In the presence of an acceptor template, (+) strand DNA transfer is efficient only with templates containing natural tRNA(Lys,)3 or the RNA PBS primer, Sequence analysis of transfer products revealed frequent errors at the transfer site with synthetic tRNA(Lys,3), not observed with natural tRNA(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 PP activity of RT also influence the efficacy of (+) strand DNA transfer.