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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Cell Line
  • Hepacivirus / chemistry
  • Hepacivirus / genetics
  • Hepacivirus / metabolism*
  • Hepatitis C / virology*
  • Humans
  • Molecular Sequence Data
  • Phosphorylation
  • RNA, Viral / chemistry
  • RNA, Viral / genetics
  • RNA, Viral / metabolism
  • Serine / genetics
  • Serine / metabolism*
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*
  • Virus Replication*


  • NS-5 protein, hepatitis C virus
  • RNA, Viral
  • Viral Nonstructural Proteins
  • Serine