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J Biol Chem, Vol. 275, Issue 20, 15474-15481, May 19, 2000

In Vitro Studies on tRNA Annealing and Reverse Transcription with Mutant HIV-1 RNA Templates*

Nancy Beerens and Ben BerkhoutDagger

From the Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam 1100 DE, The Netherlands

The human immunodeficiency virus type 1 (HIV-1) RNA genome encodes a semistable stem-loop structure, the U5-PBS hairpin, which occludes part of the tRNA primer binding site (PBS). In previous studies, we demonstrated that mutations that alter the stability of the U5-PBS hairpin inhibit virus replication. A reverse transcription defect was measured in assays with the virion-extracted RNA-tRNA complexes. We now extend these studies with in vitro synthesized wild-type and mutant RNA templates that were tested in primer annealing and reverse transcription assays. The effect of annealing temperature and the presence of the viral nucleocapsid protein on reverse transcription was analyzed for the templates with a stabilized or destabilized U5-PBS hairpin, and in reactions initiated by tRNA or DNA primers. The results of this in vitro assay are consistent with the in vivo findings, in that both tRNA annealing and initiation of reverse transcription are sensitive to stable template RNA structure. Reverse transcription initiated by a DNA primer is less hindered by secondary structure in the RNA template than tRNA primed reactions. The inhibitory effect of template structure on tRNA-primed reverse transcription is more pronounced in this in vitro assay compared with the in vivo material, indicating that the heat-annealed RNA-tRNA complex differs from the virion-extracted viral RNA-tRNA complex.

* This work was supported in part by the Netherlands Foundation for Chemical Research with financial aid from the Netherlands Organization for Scientific Research (NWO-CW).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.

Dagger To whom correspondence should be addressed: Dept. of Human Retrovirology, Academical Medical Center, University of Amsterdam, P. O. Box 22700, 1100 DE Amsterdam, The Netherlands. Tel.: 31-20-5664822; Fax: 31-20-6916531; E-mail: B.Berkhout@AMC.UVA.NL.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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