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Virology. 2016 Apr;491:27-44. doi: 10.1016/j.virol.2016.01.018. Epub 2016 Feb 10.

Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation.

Author information

1
School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia. Electronic address: nicholas.eyre@adelaide.edu.au.
2
School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia.
3
Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide, Australia.
4
Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne, Australia; The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Australia.
5
Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide, Australia; Institute for Photonics and Advanced Sensing (IPAS), University of Adelaide, Adelaide, Australia.

Abstract

Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using 'APEX2'-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα.

KEYWORDS:

Hepatitis C virus; NS5A; Phosphatidylinositol-4 kinase III alpha; Phosphorylation; Replication; Replication complex; Replication factory

PMID:
26874015
DOI:
10.1016/j.virol.2016.01.018
[Indexed for MEDLINE]
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