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J. Biol. Chem., Vol. 278, Issue 40, 38368-38375, October 3, 2003

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Steps of the Acceptor Invasion Mechanism for HIV-1 Minus Strand Strong Stop Transfer^*

Yan Chen , Mini Balakrishnan , Bernard P. Roques  and Robert A. Bambara  ¶ ||

From the Department of Biochemistry and Biophysics and the ^¶Cancer Center, University of Rochester, Rochester, New York 14642 and the Departement de Pharmacochimie Moleculaire et Structurale, U266 INSERM, URA D1500 CNRS, UER des Sciences Pharmaceutiques et Biologiques, 4, Avenue de l'Observatoire, Paris 75270 Cedex 06, France

Minus strand strong stop transfer is obligatory for completion of HIV-1 minus strand synthesis. We previously showed evidence for an acceptor invasion-initiated mechanism for minus strand transfer. In the present study, we examined the major acceptor invasion initiation site using a minus strand transfer system in vitro, containing the 97-nucleotide full-length R region. A series of DNA oligonucleotides complementary to different regions of the cDNA was designed to interfere with transfer. Oligomers covering the region around the base of the TAR hairpin were most effective in inhibiting transfer, suggesting that the hairpin base is a preferred site for acceptor invasion. The strong pausing of reverse transcriptase at the base of the TAR and the concomitant RNase H cleavages 10–19 nucleotides behind the pause site correlated with the location of the invasion site. Oligomers closer to the 5'-end of R also inhibited transfer, though less effectively, presumably by blocking strand exchange and branch migration. We propose that pausing of reverse transcriptase at the base of TAR increases RNase H cleavages, creating gaps for acceptor invasion and transfer initiation. Strand exchange then propagates by branch migration, displacing the fragmented donor RNA, including the fragment at the 5' terminus. The primer terminus switches to the acceptor, completing the transfer. Nucleocapsid (NC) protein stimulated transfer efficiency by 5–7-fold. NC enhanced RNase H cleavages close to the TAR base, creating more effective invasion sites for efficient transfer. Most likely, NC also stimulates transfer by promoting strand exchange invasion and branch migration.

Received for publication, June 2, 2003 , and in revised form, July 21, 2003.

^* This work was supported by National Institutes of Health Grant 49573. The costs of publication of this article were defrayed in part by the payment of page charges. This 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 Biochemistry and Biophysics, Box 712, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-2764; Fax: 585-271-2683; E-mail: robert_bambara{at}urmc.rochester.edu.

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