1887

Abstract

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 isolate (JFH-1) sub-genomic replicon (SGR) in Huh7 cells, but this phenotype was partially restored in Huh7.5 cells. Here we demonstrate that five additional residues, surface-exposed and proximal to V67 or P145, exhibited the same phenotype. In contrast, the analogous mutants in a genotype 3a isolate (DBN3a) SGR exhibited different phenotypes in each cell line, consistent with fundamental differences in the functions of genotypes 2 and 3 NS5A. The difference between Huh7 and Huh7.5 cells was reminiscent of the observation that cyclophilin inhibitors are more potent against HCV replication in the former and suggested a role for D1 in cyclophilin dependence. Consistent with this, all JFH-1 and DBN3a mutants exhibited increased sensitivity to cyclosporin A treatment compared to wild-type. Silencing of cyclophilin A (CypA) in Huh7 cells inhibited replication of both JFH-1 and DBN3a. However, in Huh7.5 cells CypA silencing did not inhibit JFH-1 wild-type, but abrogated replication of all the JFH-1 mutants, and both DBN3a wild-type 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. Lastly, we confirmed that JFH-1 NS5A D1 interacted with CypA . These data demonstrate both a direct involvement of NS5A D1 in cyclophilin-dependent genome replication and functional differences between genotype 2 and 3 NS5A.

Funding
This study was supported by the:
  • Medical Research Council (Award MR/S001026/1)
    • Principle Award Recipient: MarkHarris
  • Wellcome Trust (Award 096670)
    • Principle Award Recipient: MarkHarris
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2023-09-06
2024-06-23
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