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Nucleic Acids Research 2005 33(13):4285-4310; doi:10.1093/nar/gki720
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Published online 1 August 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Article

Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity

Véronique Goffin, Dominique Demonté, Caroline Vanhulle, Stéphane de Walque, Yvan de Launoit1,2, Arsène Burny, Yves Collette3 and Carine Van Lint*

Laboratoire de Virologie Moléculaire, Institut de Biologie et de Médecine Moléculaires (IBMM), Service de Chimie Biologique, Université Libre de Bruxelles Rue des Profs Jeener et Brachet 12, 6041 Gosselies, Belgium 1Faculté de Médecine, Laboratoire de Virologie Moléculaire, Université Libre de Bruxelles 808 Route de Lennik, 1070 Bruxelles, Belgium 2Institut de Biologie de Lille, Institut Pasteur de Lille, Université de Lille 1, UMR 8117 CNRS BP 447, 1 Rue Calmette, 59021 Lille Cedex, France 3INSERM U119 27 Boulevard Lei Roure, 13009 Marseille, France

*To whom correspondence should be addressed. Tel: +32 2 650 9807; Fax: +32 2 650 9800; Email: cvlint{at}ulb.ac.be

Received June 21, 2005. Accepted July 4, 2005.

We have previously identified in the pol gene of human immunodeficiency virus type 1 (HIV-1) a new positive transcriptional regulatory element (nt 4481–4982) containing recognition sites for nuclear proteins (sites B, C, D and a GC-box) [C. Van Lint, J. Ghysdael, P. Paras, Jr, A. Burny and E. Verdin (1994) J. Virol. 68, 2632–2648]. In this study, we have further physically characterized each binding site and have shown that the transcription factors Oct-1, Oct-2, PU.1, Sp1 and Sp3 interact in vitro with the pol region. Chromatin immunoprecipitation assays using HIV-infected cell lines demonstrated in the context of chromatin that Sp1, Sp3, Oct-1 and PU.1 are recruited to the HS7 region in vivo. For each site, we have identified mutations abolishing factor binding to their cognate DNA sequences without altering the underlying amino acid sequence of the integrase. By transient transfection assays, we have demonstrated the involvement of the pol binding sites in the transcriptional enhancing activity of the intragenic region. Our functional results with multimerized wild-type and mutated pol binding sites separately (i.e. in the absence of the other sites) have demonstrated that the PU.1, Sp1, Sp3 and Oct-1 transcription factors regulate the transcriptional activity of a heterologous promoter through their respective HS7 binding sites. Finally, we have investigated the physiological role of the HS7 binding sites in HIV-1 replication and have shown that these sites are important for viral infectivity.

The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors


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