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J Biol Chem, Vol. 274, Issue 7, 4412-4420, February 12, 1999
From 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 tRNALys,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
tRNALys,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 tRNALys,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 tRNALys,3. Modified
nucleoside m1A-58 of natural tRNALys,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 tRNALys,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 tRNALys,3 or the RNA PBS
primer. Sequence analysis of transfer products revealed frequent errors
at the transfer site with synthetic tRNALys,3, not observed
with natural tRNALys,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 (
Role of Post-transcriptional Modifications of Primer
tRNALys,3 in the Fidelity and Efficacy of Plus Strand
DNA Transfer during HIV-1 Reverse Transcription
,
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
) PBS of the acceptor template and the RNase H activity of RT also influence the
efficacy of (+) strand DNA transfer.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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