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

Nicholas Eyre, Rachel Hampton-Smith, Amanda Aloia, James Eddes, Kaylene Simpson, Peter Hoffmann, Michael Beard

    Research output: Contribution to journalArticlepeer-review

    22 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)27-44
    Number of pages18
    JournalVirology
    Volume491
    DOIs
    Publication statusPublished - 1 Apr 2016

    Keywords

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

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