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Antimicrobial Agents and Chemotherapy, December 2006, p. 4161-4169, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00433-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

General Catalytic Deficiency of Hepatitis C Virus RNA Polymerase with an S282T Mutation and Mutually Exclusive Resistance towards 2'-Modified Nucleotide Analogues{triangledown}

Hélène Dutartre, Cécile Bussetta, Joëlle Boretto, and Bruno Canard*

Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d'Ingénieurs de Luminy-Case 925, 163 avenue de Luminy, 13288 Marseille Cedex 9, France

Received 6 April 2006/ Returned for modification 26 May 2006/ Accepted 25 August 2006

The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is an important target for antiviral therapies. NS5B is able to initiate viral RNA synthesis de novo and then switch to a fast and processive RNA elongation synthesis mode. The nucleotide analogue 2'-C-methyl CTP (2'-C-Me-CTP) is the active metabolite of NM283, a drug currently in clinical phase II trials. The resistance mutation S282T can be selected in HCV replicon studies. Likewise, 2'-O-Me nucleotides are active both against the purified polymerase and in replicon studies. We have determined the molecular mechanism by which the S282T mutation confers resistance to 2'-modified nucleotide analogues. 2'-C-Me-CTP is no longer incorporated during the initiation step of RNA synthesis and is discriminated 21-fold during RNA elongation by the NS5B S282T mutant. Strikingly, 2'-O-methyl CTP sensitivity does not change during initiation, but the analogue is no longer incorporated during elongation. This mutually exclusive resistance mechanism suggests not only that "2'-conformer" analogues target distinct steps in RNA synthesis but also that these analogues have interesting potential in combination therapies. In addition, the presence of the S282T mutation induces a general cost in terms of polymerase efficiency that may translate to decreased viral fitness: natural nucleotides become 5- to 20-fold less efficiently incorporated into RNA by the NS5B S282T mutant. As in the case for human immunodeficiency virus, our results might provide a mechanistic basis for the rational combination of drugs for low-fitness viruses.

* Corresponding author. Mailing address: AFMB-CNRS-ESIL, Case 925, 163 avenue de Luminy, 13288 Marseille Cedex 9, France. Phone: 33-491-828644. Fax: 33-491-828646. E-mail: Bruno.Canard{at}afmb.univ-mrs.fr.

{triangledown} Published ahead of print on 25 September 2006.

Antimicrobial Agents and Chemotherapy, December 2006, p. 4161-4169, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00433-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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