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Originally published In Press as doi:10.1074/jbc.M207223200 on November 27, 2002

J. Biol. Chem., Vol. 278, Issue 10, 7902-7909, March 7, 2003
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Incorporation of Uracil into Minus Strand DNA Affects the Specificity of Plus Strand Synthesis Initiation during Lentiviral Reverse Transcription*

George J. KlarmannDagger §, Xin ChenDagger §, Thomas W. North||, and Bradley D. PrestonDagger **

From the Dagger  Eccles Institute of Human Genetics and the Department of Biochemistry, University of Utah, Salt Lake City, Utah 84112 and the || Center for Comparative Medicine, University of California, Davis, California 95616

Many retroviruses either encode dUTP pyrophosphatase (dUTPase) or package host-derived uracil DNA glycosylase as a means to limit the accumulation of uracil in DNA strands, suggesting that uracil is detrimental to one or more steps in the viral life cycle. In the present study, the effects of DNA uracilation on (-) strand DNA synthesis, RNase H activity, and (+) strand DNA synthesis were investigated in a cell-free system. This system uses the activities of purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase to convert single-stranded RNA to double-stranded DNA in a single reaction mixture. Substitution of dUTP for dTTP had no effect on (-) strand synthesis but significantly decreased yields of (+) strand DNA. Mapping of nascent (+) strand 5' ends revealed that this was due to decreased initiation from polypurine tracts with a concomitant increase in initiation at non-polypurine tract sites. Aberrant initiation correlated with a change in RNase H cleavage specificity when assayed on preformed RNA-DNA duplexes containing uracilated DNA, suggesting that appropriate "selection" of the (+) strand primer is affected. Collectively, these data suggest that accumulation of uracil in retroviral DNA may disrupt the viral life cycle by altering the specificity of (+) strand DNA synthesis initiation during reverse transcription.

* This work was supported by United States Public Health Service Grants R01 AI34834, R01 AI38755, and P30 CA42014 (to B. D. P.) and R01 AI28189 (to T. W. N.) 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.

§ These authors contributed equally to this work.

To whom correspondence should be addressed: HIV Drug Resistance Program National Cancer Institute-Frederick, P. O. Box B, Frederick, MD 21702. Tel.: 301-846-5395; Fax: 301-846-6013; E-mail: gklarmann@ncifcrf.gov.

** Present address: University of Washington Dept. of Pathology K-072 Health Sciences Bldg., Box 357705, 1959 NE Pacific St., Seattle, WA 98195-7705.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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