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J. Biol. Chem., Vol. 277, Issue 41, 38053-38061, October 11, 2002
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From the HIV-1 evolves rapidly, which is
thought to result from one or more error-prone steps in the virus life
cycle. Because HIV-1 reverse transcriptase (RT) does not possess 3'- to
5'-exonucleolytic proofreading activity and because RT has been shown
to be error-prone in cell free systems, it should be an important
contributor to the high rate of HIV-1 mutation. However, because RNA
polymerase II (pol II) synthesizes viral RNA, it might also contribute
significantly to HIV-1 mutagenesis. To assess the relative
contributions of RT and RNA pol II to HIV-1 mutagenesis, a
system was established to study the rate and nature of mutations in
HIV-1 long terminal repeats (LTRs). Owing to the unique nature of
replication at the ends of the viral genome, mutational analysis of
retroviral LTRs provides an opportunity to evaluate the relative
contribution of HIV-1 RT and RNA pol II to viral mutagenesis.
Mutational analysis was performed on both LTRs of 215 proviruses,
restricted to a single cycle of replication, employing single-stranded
conformational polymorphism and DNA sequencing allowing direct
identification of mutations in the absence of selection and within
autologous viral sequences. A total of 21 independent mutations was
identified. Ten mutations were observed in both LTRs, which could have
been introduced by either RT or RNA pol II, whereas the other eleven mutations were only present in a single LTR and could only have been
introduced by RT. This provides the first direct evidence that HIV-1 RT
contributes significantly to HIV-1 mutagenesis and is likely to be the
primary engine for HIV-1 mutagenesis. Moreover, mutations were observed
at the U3-R border, but the nature of the mutations and their frequency
differed from experiments performed using cell-free systems suggesting
that other viral and/or cellular factors contribute to fidelity at the
ends of the viral genome.
Mutational Analysis of HIV-1 Long Terminal Repeats to Explore the
Relative Contribution of Reverse Transcriptase and RNA Polymerase II to
Viral Mutagenesis*
§,
,, and§§
Department of Biochemistry and Radiation
Oncology, Eccles Institute of Human Genetics, University of Utah, Salt
Lake City, Utah 84112, the ¶ Department of Molecular Genetics,
Microbiology and Immunology, University of Medicine and Dentistry of
New Jersey-Robert Wood Johnson Medical School, Piscataway, New
Jersey 08854, and the Graduate Program in Microbiology and
Molecular Genetics, Rutgers University, New Brunswick, New Jersey
08903
*
This work was supported by Grants CA50777 and AI34834 from
the National Institutes of Health.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Genetics, Microbiology and Immunology, University of Medicine and
Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Tel.: 732-235-4588; Fax: 732-235-5223;
E-mail: doughejp@umdnj.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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