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Originally published In Press as doi:10.1074/jbc.M107441200 on December 10, 2001

J. Biol. Chem., Vol. 277, Issue 11, 8775-8789, March 15, 2002
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Upstream Stimulatory Factors Binding to an E Box Motif in the R Region of the Bovine Leukemia Virus Long Terminal Repeat Stimulates Viral Gene Expression*

Claire CalommeDagger §, Thi Liên-Anh NguyênDagger , Yvan de Launoit||**, Véronique KiermerDagger Dagger Dagger , Louis Droogmans§§¶¶, Arsène BurnyDagger , and Carine Van LintDagger ¶¶||||

From the Dagger  Université Libre de Bruxelles, Institut de Biologie et de Médecine Moléculaires, Service de Chimie Biologique, Laboratoire de Virologie Moléculaire, Rue des Profs Jeener et Brachet 12, 6041 Gosselies, Belgium, || Université Libre de Bruxelles, Faculté de Médecine, Laboratoire de Microbiologie, 808 Route de Lennik, 1070 Bruxelles, Belgium, ** Institut Pasteur de Lille, Institut de Biologie de Lille, UMR 8526 CNRS, 1 Rue Calmette BP 447, 59021 Lille Cedex, France, and §§ Université Libre de Bruxelles, Laboratoire de Microbiologie, Institut de Recherche du CERIA, 1 Avenue Emile Gryson, 1070 Bruxelles, Belgium

The bovine leukemia virus (BLV) promoter is located in its 5'-long terminal repeat and is composed of the U3, R, and U5 regions. BLV transcription is regulated by cis-acting elements located in the U3 region, including three 21-bp enhancers required for transactivation of the BLV promoter by the virus-encoded transactivator TaxBLV. In addition to the U3 cis-acting elements, both the R and U5 regions contain stimulatory sequences. To date, no transcription factor-binding site has been identified in the R region. Here sequence analysis of this region revealed the presence of a potential E box motif (5'-CACGTG-3'). By competition and supershift gel shift assays, we demonstrated that the basic helix-loop-helix transcription factors USF1 and USF2 specifically interacted with this R region E box motif. Mutations abolishing upstream stimulatory factor (USF) binding caused a reproducible decrease in basal or Tax-activated BLV promoter-driven gene expression in transient transfection assays of B-lymphoid cell lines. Cotransfection experiments showed that the USF1 and USF2a transactivators were able to act through the BLV R region E box. Taken together, these results physically and functionally characterize a USF-binding site in the R region of BLV. This E box motif located downstream of the transcription start site constitutes a new positive regulatory element involved in the transcriptional activity of the BLV promoter and could play an important role in virus replication.

* This work was supported by grants from the FNRS-Télévie, Free University of Brussels (ARC), Internationale Brachet Stiftung, CGRI-INSERM Cooperation, Région Wallonne-Commission Européenne FEDER, and Theyskens-Mineur Foundation (to C. V. L.).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.

§ Fellow of the FNRS-Télévie.

Fellow of the Belgian "Fonds pour la Recherche dans l'Industrie et l'Agriculture (FRIA)."

Dagger Dagger Present address: The Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94141.

¶¶ "Chercheurs Qualifiés" of the "Fonds National de la Recherche Scientifique" (Belgium).

|||| To whom correspondence should be addressed: Université Libre de Bruxelles, Institut de Biologie et de Médecine Moléculaires, Service de Chimie Biologique, Laboratoire de Virologie Moléculaire, Rue des Profs Jeener et Brachet, 12, 6041 Gosselies, Belgium. Tel.: 32-2-650-9807; Fax: 32-2-650-9800; E-mail: cvlint@dbm.ulb.ac.be.

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


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