Development of novel infectious clones of genotype 1 hepatitis C virus

Abstract

Background & Aims: Hepatitis C virus (HCV) research has been hampered by the inability to culture HCV isolates in vitro except for the genotype 2a JFH1. Thus, it is important to develop cell culture-infectious clones for various HCV genotypes and to study cell culture-adaptive mutations which would enable development of infectious HCV clones. Among cell culture-adaptive mutations, S2204I robustly enhances HCV RNA replication in most HCV genotypes but the relevant mechanism is still unclear. Recent studies identified four cell culture-adaptive mutations F1464L/A1672S/D2979G/ Y2981F (LSGF) that permit many HCV isolates to be further adapted in cell culture. In this study, LSGF mutations were used as the first step for developing genotype 1 infectious clones and we also investigated the effect of S2204I mutation on viral RNA replication.

Method: Multiple cell culture-adaptive mutations including LSGF were introduced into genotype 1a and 1b HCV sequences. We also generated various recombinant chimeras with or without S2204I mutation. And we generated genotype 1a constructs including substitutions at position 2204 (threonine and serine) which were found in the chimpanzee-infection experiment. Huh7.5 cells were transfected with in vitro-transcribed viral RNA and HCV RNA replication was evaluated by Gaussia luciferase reporter assay.

Results: LSGF mutations did not confer the RNA replication ability of H77C and H77S.3 (genotype 1a). Con1 (genotype 1b) that was introduced with multiple cell culture-adaptive mutations including LSGF showed a low RNA replication level. The RNA replication of H77S-based JFH1 chimeras in transfected cells was impaired regardless of S2204I mutation. RNA replication level of H77S.3 with either threonine or serine at position 2204 was similar to each other.

Conclusion: LSGF mutations did not increase RNA replication capacities of H77C, H77S.3 and Con1. Capability of S2204I mutation in enhancing HCV RNA replication was not dependent on background genotypes. Serine and threonine at position 2204 supported HCV RNA replication similarly in in-vitro transfection experiments.