Effect of the reversion of NS5A S2204I mutation on the replication of hepatitis C virus

Abstract

Background & Aims: The nonstructural protein 5A (NS5A) of hepatitis C virus is a phosphoprotein that is required for both RNA replication and virion assembly. Differential phosphorylation has been proposed as a molecular switch that modulates the two functions of NS5A protein. Efficient replication of H77 (genotype 1a) in Huh7.5 cells requires five cell culture-adaptive mutations, including K2040R andS2204I in NS5A protein. However, replication of JFH1 (genotype 2a) in cell culture system does not require any cell culture-adaptive mutations. Therefore in this study, we tested whether the reversion of K2040R and S2204I cell culture-adaptive mutations of H77S NS5A to wild-type sequence have any effect on viral RNA replication in diverse genotypic backgrounds.Method: We generated various JFH1-based H77 NS5A chimeras, including both K2040R and S2204I cell culture-adaptive mutations (JFH1/H5A), single amino acid substitution mutations (JFH1/H5A/RK andJFH1/H5A/IS), and a wild-type sequence (JFH1/H5A/RKIS). Huh-7.5 cells were transfected with in-vitro transcribed viral RNA from the respective recombinant plasmid DNA and viral replication was evaluated by Gaussia luciferase reporter assay.Results: The efficiency of viral replication was severely impaired in cells transfected with JFH1/H5A. The reversion of S2204I to a wild-type sequence (JFH/H5A/IS) restored viral RNA replication capacity. Similar levels of replication efficiency was observed for the constructs bearing both R2040K and I2204S substitution mutations (JFH1/H5A/RKIS). However, R2040K substitution alone (JFH1/H5A/RK) did not affect the viral replication rate. Focus-forming assay showed that no infectious virus particles were released from cells transfected with the JFH1/H5A and JFH1/H5A/RK. In contrast, infectious virus particles were efficiently released by cells transfected with the JFH1/H5A/IS and JFH1/H5A/RKIS, the titer of which was comparable to that of wild-type JFH1. Consistent with these results, immunoblot analysis demonstrated no significant differences in the expression levels of NS5A protein in cells transfected with JFH1/H5A/IS, JFH1/H5A/RKIS.Conclusion: S2204I cell culture-adaptive mutation, conferring high viral replication in H77 strains, had negative effect in the context of JFH1. This result suggests that phosphorylation of serine residue at position 2204 in NS5A protein is important for efficient replication of JFH1 RNA