Yin, C, Goonawardane, N, Stewart, H et al. (1 more author) (2018) A role for domain I of the hepatitis C virus NS5A protein in virus assembly. PLoS Pathogens, 14 (1). e1006834. ISSN 1553-7366
Abstract
The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and assembly. NS5A comprises three domains, of these domain I is believed to be involved exclusively in genome replication. In contrast, domains II and III are required for the production of infectious virus particles and are largely dispensable for genome replication. Domain I is highly conserved between HCV and related hepaciviruses, and is highly structured, exhibiting different dimeric conformations. To investigate the functions of domain I in more detail, we conducted a mutagenic study of 12 absolutely conserved and surface-exposed residues within the context of a JFH-1-derived sub-genomic replicon and infectious virus. Whilst most of these abrogated genome replication, three mutants (P35A, V67A and P145A) retained the ability to replicate but showed defects in virus assembly. P35A exhibited a modest reduction in infectivity, however V67A and P145A produced no infectious virus. Using a combination of density gradient fractionation, biochemical analysis and high resolution confocal microscopy we demonstrate that V67A and P145A disrupted the localisation of NS5A to lipid droplets. In addition, the localisation and size of lipid droplets in cells infected with these two mutants were perturbed compared to wildtype HCV. Biophysical analysis revealed that V67A and P145A abrogated the ability of purified domain I to dimerize and resulted in an increased affinity of binding to HCV 3’UTR RNA. Taken together, we propose that domain I of NS5A plays multiple roles in assembly, binding nascent genomic RNA and transporting it to lipid droplets where it is transferred to Core. Domain I also contributes to a change in lipid droplet morphology, increasing their size. This study reveals novel functions of NS5A domain I in assembly of infectious HCV and provides new perspectives on the virus lifecycle.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | (c) 2018 Yin et al. This is an open access article distributedunder the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. (https://creativecommons.org/licenses/by/4.0/) |
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) |
Depositing User: | Symplectic Publications |
Date Deposited: | 23 Jan 2018 15:52 |
Last Modified: | 25 Jun 2023 21:13 |
Status: | Published |
Publisher: | Public Library of Science |
Identification Number: | 10.1371/journal.ppat.1006834 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:126499 |