title: Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation.
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.
authors.affiliation: 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.
authors.forename: Nicholas S
authors.initials: NS
authors.lastname: Eyre
authors.affiliation: School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia.
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Hampton-Smith
authors.affiliation: School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Aloia
authors.affiliation: Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide, Australia.
authors.forename: James S
authors.initials: JS
authors.lastname: Eddes
authors.affiliation: Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne, Australia; The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Australia.
authors.forename: Kaylene J
authors.initials: KJ
authors.lastname: Simpson
authors.affiliation: Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide, Australia; Institute for Photonics and Advanced Sensing (IPAS), University of Adelaide, Adelaide, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Hoffmann
authors.affiliation: School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Beard
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002460:Cell Line; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D012367:RNA, Viral; D012694:Serine; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: Hepatitis C virus; NS5A; Phosphatidylinositol-4 kinase III alpha; Phosphorylation; Replication; Replication complex; Replication factory