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

• Published in: The Journal of biological chemistry Vol. 274; no. 7; pp. 4412 - 4420
• Main Authors: Auxilien, S, Keith, G, Le Grice, S F, Darlix, J L
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 12-02-1999
• Subjects: AIDS/HIV; Base Sequence; DNA, Viral - metabolism; HIV Reverse Transcriptase - metabolism; HIV-1 - physiology; Molecular Sequence Data; Nucleic Acid Conformation; Ribonuclease H - metabolism; RNA Processing, Post-Transcriptional; RNA, Transfer, Lys - metabolism; RNA, Viral - metabolism; Templates, Genetic; Transcription, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.7.4412

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 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 H activity of RT also
influence the efficacy of (+) strand DNA transfer.