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J. Biol. Chem., Vol. 279, Issue 24, 25489-25496, June 11, 2004

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A Loss of Viral Replicative Capacity Correlates with Altered DNA Polymerization Kinetics by the Human Immunodeficiency Virus Reverse Transcriptase Bearing the K65R and L74V Dideoxynucleoside Resistance Substitutions^*

Jérôme Deval¶, Jean-Marc Navarro||, Boulbaba Selmi**, Jérôme Courcambeck, Joëlle Boretto, Philippe Halfon, Sarah Garrido-Urbani||, Josephine Sire||, and Bruno Canard

From the CNRS and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, École Supérieure d'Ingénieurs de Luminy-Case 925, ^||INSERM U-372, and /Genosciences, Département des Sciences Chimiques, 163 avenue de Luminy, 13288 Marseille cedex 9, Marseille, France

Mechanisms governing viral replicative capacity are poorly understood at the biochemical level. Human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) K65R or L74V substitutions confer viral resistance to 2',3'-dideoxyinosine (ddI) in vivo. The two substitutions never occur together, and L74V is frequently found in patients receiving ddI, while K65R is not. Here we show that recombinant viruses carrying K65R and K65R/L74V display the same resistance level to ddI (about 9.5-fold) relative to wild type. Consistent with this result, purified HIV-1 RT carrying K65R RT or K65R/L74V substitutions exhibits an 8-fold resistance to ddATP as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. Resistance is due to a selective decrease of the catalytic rate constant k_pol: 22-fold (from 7.2 to 0.33 s^-1) for K65R RT and 84-fold (from 7.2 to 0.086 s^-1) for K65R/L74V RT. However, the K65R/L74V virus replication capacity is severely impaired relative to that of wild-type virus. This loss of viral fitness is correlated to a poor ability of K65R/L74V RT to use natural nucleotides relative to wild-type RT: 15% that of wild-type RT for dATP, 36% for dGTP, 50% for dTTP, and 25% for dCTP. The order of incorporation efficiency is wild-type RT > L74V RT > K65R RT > K65R/L74V RT. Processivity of DNA synthesis remains unaffected. These results explain why the two mutations do not combine in the clinic and might give a mechanism for a decreased viral fitness at the molecular level.

Received for publication, December 10, 2003 , and in revised form, February 2, 2004.

^* This investigation was supported in part by the Agence Nationale de Recherche sur le SIDA (ANRS) and Ensemble Contre le SIDA (ECS). 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.

Both authors contributed equally to this work.

^¶ Supported by a predoctoral fellowship from the ANRS.

^** Supported by a postdoctoral fellowship from ECS.

To whom correspondence should be addressed. Tel.: 33-491-82-86-44; Fax: 33-491-82-86-46; E-mail: bruno{at}afmb.cnrs-mrs.fr.

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