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J. Biol. Chem., Vol. 281, Issue 17, 11736-11743, April 28, 2006

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Nucleotide Excision Repair and Template-independent Addition by HIV-1 Reverse Transcriptase in the Presence of Nucleocapsid Protein^*

Carole Bampi, Arkadiusz Bibillo, Michaela Wendeler, Gilles Divita^¶, Robert J. Gorelick^||, Stuart F. J. Le Grice, and Jean-Luc Darlix¹

From the LaboRetro, Unité de Virologie Humaine, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, Institut Fédératif de Recherche 128, 69364 Lyon Cedex 07, France, HIV-Drug Resistance Program, NCI, National Institutes of Health, Frederick, Maryland 21702, ^¶Centre de Recherche de Biochimie Macromoléculaire-CNRS-Formation de Recherche en Evolution-2593, 1919 Route de Mende, 36293 Montpellier Cedex 05, France, and ^||AIDS Vaccine Program, Science Applications International Corporation-Frederick, Inc., NCI, National Institutes of Health, Frederick, Maryland 21702

During HIV replication, reverse transcriptase (RT), assisted by the nucleocapsid protein (NC), converts the genomic RNA into proviral DNA. This process appears to be the major source of genetic variability, as RT can misincorporate nucleotides during minus and plus strand DNA synthesis. To investigate nucleotide addition or substitution by RT, we set up in vitro models containing HIV-1 RNA, cDNA, NC, and various RTs. We used the wild type RT and azidothymidine- and didanosine-resistant RTs, because they represent the major forms of resistant RTs selected in patients undergoing therapies. Results show that all RTs can add nucleotides in a non-template fashion at the cDNA 3'-end, a reaction stimulated by NC. Nucleotide substitutions were examined using in vitro systems where 3'-mutated cDNAs were extended by RT on an HIV-1 RNA template. With NC, RT extension of the mutated cDNAs was efficient, and surprisingly, mutations were frequently corrected. These results suggest for the first time that RT has excision-repair activity that is triggered by NC. Chaperoning of RT by NC might be explained by the fact that NC stabilizes an RT-DNA binary complex. In conclusion, RT-NC interactions appear to play critical roles in HIV-1 variability.

Received for publication, January 11, 2006 , and in revised form, February 13, 2006.

^* This work was supported by INSERM, Agence Nationale de Recherches sur le SiDA, Fondation pour la Recherche Médicale, and Europe (Targeting Replication and Integration of HIV). This research was supported in part by the Intramural Research Program of the National Institutes of Health (NO1-CO 12400) (to R. J. G.), National Cancer Institute, and the Center for Cancer Research. 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.

¹ To whom correspondence should be addressed. Tel.: 33-4-72-72-81-69; Fax: 33-4-72-72-80-80; E-mail: Jean-Luc.Darlix{at}ens-lyon.fr.

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