Mutational analysis reveals a novel role for hepatitis C virus NS5A domain I in cyclophilin-dependent genome replication

The hepatitis C virus (HCV) NS5A protein is comprised of three domains (D1-3). Previously, we observed that two alanine substitutions in D1 (V67A, P145A) abrogated replication of a genotype 2a (JFH-1 isolate) sub-genomic replicon (SGR) in Huh7 cells, but this phenotype was partially restored in Huh7.5 cells. To investigate the mechanism of this difference we extended this analysis to demonstrate that 5 additional residues, surface exposed and proximal to either V67 or P145, exhibited the same phenotype. In contrast, these mutants in a genotype 3a (DBN3a isolate) SGR retained their phenotype in each cell line. The difference between Huh7 and Huh7.5 cells was reminiscent of the observation that cyclophilin (Cyp) inhibitors are more potent against HCV replication in the former and suggested a role for D1 in Cyp dependence. Consistent with this, all JFH-1 and DBN3a mutants exhibited increased sensitivity to cyclosporin A treatment compared to wildtype. Silencing of CypA in Huh7 cells inhibited replication of both JFH-1 and DBN3a. However, in Huh7.5 cells CypA silencing did not inhibit JFH-1 wildtype, but abrogated replication of all the JFH-1 mutants, and both DBN3a wildtype and all mutants. CypB silencing in Huh7 cells had no effect on DBN3a, but abrogated replication of JFH-1. CypB silencing in Huh7.5 cells had no effect on either SGR. These data demonstrate both a direct involvement of NS5A D1 in Cyp-dependent genome replication and functional differences between genotype 2 and 3 NS5A. Lastly, we confirmed that JFH-1 NS5A D1 interacted with CypA in vitro.