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Originally published In Press as doi:10.1074/jbc.M108972200 on December 13, 2001

J. Biol. Chem., Vol. 277, Issue 8, 5995-6004, February 22, 2002
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In Vitro Evidence for a Long Range Pseudoknot in the 5'-Untranslated and Matrix Coding Regions of HIV-1 Genomic RNA*

Jean-Christophe PaillartDagger §, Eugene SkripkinDagger , Bernard EhresmannDagger , Chantal EhresmannDagger , and Roland MarquetDagger

From the Dagger  UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, Strasbourg F-67084, France

The 5'-untranslated leader region of human immunodeficiency virus type 1 (HIV-1) RNA contains multiple signals that control distinct steps of the viral replication cycle such as transcription, reverse transcription, genomic RNA dimerization, splicing, and packaging. It is likely that fine tuned coordinated regulation of these functions is achieved through specific RNA-protein and RNA-RNA interactions. In a search for cis-acting elements important for the tertiary structure of the 5'-untranslated region of HIV-1 genomic RNA, we identified, by ladder selection experiments, a short stretch of nucleotides directly downstream of the poly(A) signal that interacts with a nucleotide sequence located in the matrix region. Confirmation of the sequence of the interacting sites was obtained by partial or complete inhibition of this interaction by antisense oligonucleotides and by nucleotide substitutions. In the wild type RNA, this long range interaction was intramolecular, since no intermolecular RNA association was detected by gel electrophoresis with an RNA mutated in the dimerization initiation site and containing both sequences involved in the tertiary interaction. Moreover, the functional importance of this interaction is supported by its conservation in all HIV-1 isolates as well as in HIV-2 and simian immunodeficiency virus. Our results raise the possibility that this long range RNA-RNA interaction might be involved in the full-length genomic RNA selection during packaging, repression of the 5' polyadenylation signal, and/or splicing regulation.

* This work was supported by grants from the "Agence Nationale de Recherches sur le SIDA" and a "Jeunes Equipes" grant from the Center National de la Recherche Scientifique (to R. M.).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: UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67094 Strasbourg cedex, France. Tel.: 33-3-88-41-70-91; Fax: 33-3-88-60-22-18; E-mail: JC.Paillart@ibmc.u-strasbg.fr.

Present address: Memorial Sloan-Kettering Cancer Center, Box 557, York Ave., New York, NY 10021.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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