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J. Biol. Chem., Vol. 279, Issue 29, 29963-29973, July 16, 2004

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Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA^*

Delphine Ropers, Lilia Ayadi¶, Renata Gattoni||, Sandrine Jacquenet**, Laurence Damier, Christiane Branlant, and James Stévenin||

From the Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR CNRS 7567, Université Henri Poincaré Nancy 1, Boulevard des Aiguillettes, BP239, 54506 Vandoeuvre-lès-Nancy Cedex and ^||IGBMC, UMR CNRS-INSERM-ULP 7104, 1 Rue Laurent Fries, BP10142, Parc d'Innovation, 67404 Illkirch Cedex, France

Splicing is a crucial step for human immunodeficiency virus, type 1 (HIV-1) multiplication; eight acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs. The effects of SR proteins have only been investigated on a limited number of HIV-1 splicing sites by using small HIV-1 RNA pieces. To understand how SR proteins influence the use of HIV-1 splicing sites, we tested the effects of overproduction of individual SR proteins in HeLa cells on the splicing pattern of an HIV-1 RNA that contained all the splicing sites. The steady state levels of the HIV-1 mRNAs produced were quantified by reverse transcriptase-PCR. For interpretation of the data, transcripts containing one or several of the HIV-1 acceptor sites were spliced in vitro in the presence or the absence of one of the tested SR proteins. Both in vivo and in vitro, acceptor sites A2 and A3 were found to be strongly and specifically regulated by SR proteins. ASF/SF2 strongly activates site A2 and to a lesser extent site A1. As a result, upon ASF/SF2 overexpression, the vpr mRNA steady state level is specifically increased. SC35 and SRp40, but not 9G8, strongly activate site A3, and their overexpression ex vivo induces a dramatic accumulation of the tat mRNA, to the detriment of most of the other viral mRNAs. Here we showed by Western blot analysis that the Nef protein synthesis is strongly decreased by overexpression of SC35, SRp40, and ASF/SF2. Finally, activation by ASF/SF2 and 9G8 was found to be independent of the RS domain. This is the first investigation of the effects of variations of individual SR protein concentrations that is performed ex vivo on an RNA containing a complex set of splicing sites.

Received for publication, April 22, 2004

^* This work was supported by the ANRS, the CNRS, the French Ministère de l' Education Nationale et de la Recherche, the INSERM, SIDACTION, and the Région Lorraine. 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.

Fellow of the French Ministère de la Jeunesse, de l'Education Nationale et de la Recherche. Present address: Unité de Recherche INRIA Rhône Alpes, Montbonnot, 38334 Saint Ismier Cedex, France.

^¶ Fellow of the French Agence Nationale de Recherche sur le SIDA.

^** Present address: Médecine et Thérapeutique Moléculaire (INSERM CIC9501), 54500 Vandoeuvre-lès Nancy, France.

Fellow of the SIDACTION Foundation. Present address: Unité des Agents Antibactériens, Institut Pasteur, 25-28 Rue du Dr Roux, 75015 Paris, France.

To whom correspondence should be addressed: UMR CNRS-UHP 7567, Faculté des Sciences, BP239, 54506 Vandoeuvre-lès-Nancy Cedex, France. Tel.: 3-83-68-43-03; Fax: 3-83-68-43-07; E-mail: Christiane.Branlant{at}maem.uhp-nancy.fr.

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