General catalytic deficiency of hepatitis C virus RNA polymerase with an S282T mutation and mutually exclusive resistance towards 2'-modified nucleotide analogues.
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.
Dutartre
Hélène
H
AFMB-CNRS-ESIL, Case 925, 163 avenue de Luminy, 13288 Marseille Cedex 9, France.
Bussetta
Cécile
C
Boretto
Joëlle
J
Canard
Bruno
B
D001665:Binding Sites; D003562:Cytidine / Q000031:analogs & derivatives / Q000235:genetics; D024882:Drug Resistance, Viral; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009711:Nucleotides / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*
D009711:Nucleotides; D012367:RNA, Viral; C513402:2'-C-methylcytidine; D003562:Cytidine; D012324:RNA-Dependent RNA Polymerase