Construction and characterization of Genotype-3 hepatitis C virus replicon revealed critical genotype-3-specific polymorphism for drug resistance and viral fitness.

Hepatitis C virus (HCV), a major causative agent of chronic hepatitis, is a positive-stranded RNA virus and has a high degree of genetic diversity due to its error-prone RNA-dependent RNA polymerase. Development of direct-acting antiviral agents (DAAs) has greatly improved the therapeutic outcome of chronic hepatitis C patients. However, naturally existing resistance-associated variants (RAVs) or occurrence of resistance-associated substitutions (RASs) in the HCV genome may impose a challenge to the long-term success of the DAA-based therapies. Genotype-3 HCV is the most difficult genotype to treat by DAAs, but the underlying molecular mechanisms remain to be explored. Here we developed a novel genotype-3a subgenomic replicon PR87A7 by screening a HCV cDNA pool amplified from a patient serum RNA. PR87A7 replicon displayed strong resistance to anti-NS3 DAAs, mainly owing to a genotype-3-specific polymorphism 168Q in NS3. Introduction of NS3 168Q into a genotype-2a JFH1 strain rendered resistance to anti-NS3 DAAs while greatly diminished the viral replication, and yet this fitness defect can be rescued by additional genotype-3-specific polymorphism. In conclusion, we developed a novel genotype-3a subgenomic replicon by a functional screening approach, and revealed genotype-3-specfic amino acid residues that confer resistance to anti-NS3 DAAs while retaining viral fitness.