Identification of hepatitis C virus genotype 2a replicon variants with reduced susceptibility to ribavirin

Ribavirin (RBV), a nucleoside analogue, is used in the treatment of hepatitis C virus (HCV) infection in combination with interferons. However, potential mechanisms of RBV resistance during HCV replication remain poorly understood. Serial passage of cells harboring HCV genotype 2a replicon in the presence of RBV resulted in the reduced susceptibility of the replicon to RBV. Transfection of fresh cells with RNA from RBV-resistant replicon cells demonstrated that the RBV resistance observed is largely replicon-derived. Four major amino acid substitutions: T1134S in NS3, P1969S in NS4B, V2405A in NS5A, and Y2471H in NS5B region, were identified. Site-directed mutagenesis of these mutations into the replicon indicated that Y2471H plays a role in the reduced susceptibility to RBV and leads to decrease in replication fitness. The results, in addition to analysis of sequence database, suggest that HCV variants with reduced susceptibility to RBV identified are preferential to genotype 2a.