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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.
Highlights
Hepatitis C virus (HCV) is a globally prevalent human pathogen and a leading cause of liver cirrhosis and hepatocellular carcinoma, accounting for up to 500 000 related deaths per year [1]
As we had previously demonstrated that S247 and T249 mutations had no phenotype in either SGR or infectious virus [26], we focussed on the three threonine residues at the
We present here the results of a mutagenic analysis of potential sites of threonine phosphorylation in low complexity sequences (LCS) I of HCV non-structural 5A (NS5A)
Summary
Hepatitis C virus (HCV) is a globally prevalent human pathogen and a leading cause of liver cirrhosis and hepatocellular carcinoma, accounting for up to 500 000 related deaths per year [1]. HCV has a positive-strand RNA genome (~9.6 kb in length) that encodes a large polyprotein (~3000 amino acids) flanked by 5¢ and 3¢-UTRs. The polyprotein is processed by viral and host proteases into 10 viral proteins: core, envelope glycoproteins E1 and E2, the viroporin p7, and the non-structural (NS) proteins NS2-NS5B [4, 5]. The structural proteins, core, E1 and E2, are essential components of the HCV virion and necessary for viral assembly, entry and fusion [6, 7], whereas p7 and NS2 are required for assembly of, but do not form part of, infectious virus particles. Numerous prior studies demonstrated that HCV (mediated in particular by NS4B and NS5A) induces extensive rearrangement of the endoplasmic reticulum to create a membranous web, which is essential for viral RNA replication [7,8,9]
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