Goonawardane, N, Ross-Thriepland, D and Harris, M orcid.org/0000-0002-9821-1003 (2018) Regulation of hepatitis C virus replication via threonine phosphorylation of the NS5A protein. The Journal of General Virology, 99 (1). pp. 62-72. ISSN 0022-1317
Abstract
The hepatitis C virus non-structural 5A (NS5A) protein is highly phosphorylated and plays roles in both virus genome replication and assembly of infectious virus particles. NS5A comprises three domains separated by low complexity sequences (LCS). Mass spectrometry analysis of NS5A revealed the existence of a singly phosphorylated tryptic peptide corresponding to the end of LCS I and the beginning of domain II that contained a number of potential phosphorylatable residues (serines and threonines). Here we use a mutagenic approach to investigate the potential role of three of these threonine residues. Phosphomimetic mutations of two of these (T242E and T244E) resulted in significant reductions in virus genome replication and the production of infectious virus, suggesting that the phosphorylation of these residues negatively regulated virus RNA synthesis. Mutation of T245 had no effect, however when T245E was combined with the other two phosphomimetic mutations (TripleE) the inhibitory effect on replication was less pronounced. Effects of the mutations on the ratio of basally/hyperphosphorylated NS5A, together with the apparent molecular weight of the basally phosphorylated species were also observed. Lastly, two of the mutations (T245A and TripleE) resulted in a perinuclear restricted localization of NS5A. These data add further complexity to NS5A phosphorylation and suggest that this analysis be extended outwith the serine-rich cluster within LCS I.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | (c) 2018 The Authors. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. |
Keywords: | Huh7 cells, phosphorylation, RNA replication, hepatitis C virus, NS5A, infectivity |
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 |
Depositing User: | Symplectic Publications |
Date Deposited: | 27 Nov 2017 15:24 |
Last Modified: | 23 Jun 2023 22:40 |
Status: | Published |
Publisher: | Microbiology Society |
Identification Number: | 10.1099/jgv.0.000975 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:124530 |