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

Journal Article · Fri Apr 15 04:00:00 EDT 2016 · Virology

DOI:https://doi.org/10.1016/J.VIROL.2016.01.018· OSTI ID:22581682

Hampton-Smith, Rachel J.; Aloia, Amanda L. ^[1]; Eddes, James S. ^[2]; Simpson, Kaylene J. ^[3]; Hoffmann, Peter ^[2]; Beard, Michael R. ^[1]

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1. School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide (Australia)
2. Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide (Australia)
3. Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne (Australia)

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. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.

• OSTI ID: 22581682
• Journal Information: Virology, Journal Name: Virology Vol. 491; ISSN VIRLAX; ISSN 0042-6822
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
ANTIBODIES
ELECTRON MICROSCOPY
HEPATITIS
MEMBRANES
MORPHOLOGY
MUTANTS
MUTATIONS
PHOSPHORYLATION
PHOSPHOTRANSFERASES
RNA
SERINE
VIRUSES