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J. Biol. Chem., Vol. 279, Issue 46, 48397-48403, November 12, 2004

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First Snapshots of the HIV-1 RNA Structure in Infected Cells and in Virions^*

Jean-Christophe Paillart, Markus Dettenhofer¶||, Xiao-fang Yu¶, Chantal Ehresmann, Bernard Ehresmann, and Roland Marquet

From the Unité Propre de Recherche 9002 du CNRS conventionnée à l'Université Louis Pasteur, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg cedex, France and the ^¶Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205

With the increasing interest of RNAs in regulating a range of cell biological processes, very little is known about the structure of RNAs in tissue culture cells. We focused on the 5'-untranslated region of the human immunodeficiency virus type 1 RNA genome, a highly conserved RNA region, which contains structural domains that regulate key steps in the viral replication cycle. Up until now, structural information only came from in vitro studies. Here, we developed chemical modification assays to test nucleotide accessibility directly in infected cells and viral particles, thus circumventing possible biases and artifacts linked to in vitro assays. The secondary structure of the 5'-untranslated region in infected cells points to the existence of the various stem-loop motifs associated to distinct functions, proposed from in vitro probing, mutagenesis, and phylogeny. However, compared with in vitro data, subtle differences were observed in the dimerization initiation site hairpin, and none of the proposed long range interactions were observed between the functional domains. Moreover, no global RNA rearrangement was observed; structural differences between infected cells and viral particles were limited to the primer binding site, which became protected against chemical modification upon tRNA₃ ^Lys annealing in virions and to the main packaging signal. In addition, our data suggested that the genomic RNA could already dimerize in the cytoplasm of infected cells. Taken together, our results provided the first analysis of the dynamic of RNA structure of the human immunodeficiency virus type 1 RNA genome during virus assembly ex vivo.

Received for publication, July 22, 2004 , and in revised form, September 7, 2004.

^* This work was supported by grants from the Agence Nationale de Recherches sur le SIDA (ANRS) (to C. E. and R. M.). 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.

^|| Present address: Dept. of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115.

To whom correspondence should be addressed: UPR 9002-CNRS, Institut de Biologie Moléculaire et Cellulaire, 15, rue René Descartes, 67084 Strasbourg cedex, France. Tel.: 330-3-88-41-70-35; Fax: 330-3-88-60-22-18; E-mail: jc.paillart{at}ibmc.u-strasbg.fr.

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