Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2

► The cell culture system with recombinant HCV replaced with NS5A was developed. ► Recombinant HCV with NS5A of genotype 2 strains showed efficient virus assembly. ► Mutations at the C-terminus of NS5A were responsible for the efficient virus assembly. ► Recombinant HCV with genotype 1 strains were susceptible to the NS5A inhibitor. ► Recombinant HCV with genotype 2 strains were resistant to the NS5A inhibitor. Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays multiple and diverse roles in the viral lifecycle, and is currently recognized as a novel target for anti-viral therapy. To establish an HCV cell culture system with NS5A of various strains, recombinant viruses were generated by replacing NS5A of strain JFH-1 with those of strains of genotypes 1 (H77; 1a and Con1; 1b) and 2 (J6CF; 2a and MA; 2b). All these recombinant viruses were capable of replication and infectious virus production. The replacement of JFH-1 NS5A with those of genotype 1 strains resulted in similar or slightly reduced virus production, whereas replacement with those of genotype 2 strains enhanced virus production as compared with JFH-1 wild-type. A single cycle virus production assay with a CD81-negative cell line revealed that the efficient virus production elicited by replacement with genotype 2 strains depended on enhanced viral assembly, and that substitutions in the C-terminus of NS5A were responsible for this phenotype. Pulse-chase assays revealed that these substitutions in the C-terminus of NS5A were possibly associated with accelerated cleavage kinetics at the NS5A–NS5B site. Using this cell culture system with NS5A-substituted recombinant viruses, the anti-viral effects of an NS5A inhibitor were then examined. A 300- to 1000-fold difference in susceptibility to the inhibitor was found between strains of genotypes 1 and 2. This system will facilitate not only a better understanding of strain-specific roles of NS5A in the HCV lifecycle, but also enable the evaluation of genotype and strain dependency of NS5A inhibitors.