Chen, S. and Harris, M. orcid.org/0000-0002-9821-1003 (Cover date: 2023) Mutational analysis reveals a novel role for hepatitis C virus NS5A domain I in cyclophilin-dependent genome replication. The Journal of General Virology, 104 (9). ISSN 0022-1317
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 in vitro. These data demonstrate both a direct involvement of NS5A D1 in cyclophilin-dependent genome replication and functional differences between genotype 2 and 3 NS5A.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2023 The Authors. 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. |
Keywords: | hepatitis C virus, genotype, non-structural 5A protein (NS5A), genome replication, sub-genomic replicon, cyclophilin, cyclosporin |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Virology 1 (Leeds) |
Funding Information: | Funder Grant number Wellcome Trust 096670/Z/11/Z MRC (Medical Research Council) MR/S001026/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 19 Sep 2023 12:12 |
Last Modified: | 19 Sep 2023 12:12 |
Published Version: | https://www.microbiologyresearch.org/content/journ... |
Status: | Published |
Publisher: | Microbiology Society |
Identification Number: | 10.1099/jgv.0.001886 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:203439 |