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J Biol Chem, Vol. 274, Issue 7, 4412-4420, February 12, 1999

Role of Post-transcriptional Modifications of Primer tRNA^Lys,3 in the Fidelity and Efficacy of Plus Strand DNA Transfer during HIV-1 Reverse Transcription

Sylvie Auxilien, Gérard Keith^§, Stuart F. J. Le Grice^¶, and Jean-Luc Darlix

From  LaboRetro ENS, INSERM U412, 46 allée d'Italie, 69364 Lyon cedex 07, France, ^§ Institut de Biologie Moleculaire et Cellulaire, CNRS UPR 9002, 15 rue Descartes, 67084 Strasbourg cedex, France, and the ^¶ Division of Infectious Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

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 tRNA^Lys,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 m¹A-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 (ms²t⁶A-37) in the anticodon loop. m¹A-58 is almost always transcribed into A, whereas other modified nucleosides are transcribed correctly, except for m⁷G-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 m¹A-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.

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