Phosphorylation of Serine 235 of the Hepatitis C Virus Non-Structural Protein NS5A by Multiple Kinases

PLoS One. 2016 Nov 22;11(11):e0166763. doi: 10.1371/journal.pone.0166763. eCollection 2016.

Abstract

Phosphorylation at serine 235 (S235) of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays a critical role in the viral life cycle. For medical and virological interests, we exploited the HEK293T kidney cells to test 3 candidate protein kinases on NS5A S235 phosphorylation. Inhibitors that inhibit casein kinase I α (CKIα), polo-like kinase I (PlKI) or calmodulin-dependent kinase II (CaMKII) all reduced NS5A S235 phosphorylation. CKIα was studied previously and PlKI had severe cytotoxicity, thus CaMKII was selected for validation in the Huh7.5.1 liver cells. In the HCV (J6/JFH1)-infected Huh7.5.1 cells, CaMKII inhibitor reduced NS5A S235 phosphorylation and HCV RNA levels without apparent cytotoxicity. RT-PCR analysis showed expression of CaMKII γ and δ isoforms in the Huh7.5.1 cells. Both CaMKII γ and δ directly phosphorylated NS5A S235 in vitro. CaMKII γ or δ single knockdown did not affect NS5A S235 phosphorylation but elevated the HCV RNA levels in the infected cells. CKIα plus CaMKII (γ or δ) double knockdown reduced NS5A S235 phosphorylation and reduced HCV RNA levels; however, the HCV RNA levels were higher than those in the infected cells with CKIα single knockdown. We conclude that CKIα-mediated NS5A S235 phosphorylation is critical for HCV replication. CaMKII γ and δ may have negative roles in the HCV life cycle.

MeSH terms

  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / genetics
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism*
  • Casein Kinase Ialpha / genetics
  • Casein Kinase Ialpha / metabolism*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Line, Tumor
  • Gene Knockdown Techniques
  • Hepacivirus / physiology*
  • Humans
  • Phosphorylation / physiology
  • Polo-Like Kinase 1
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism
  • Serine / genetics
  • Serine / metabolism
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*
  • Virus Replication / physiology*

Substances

  • Cell Cycle Proteins
  • Proto-Oncogene Proteins
  • Viral Nonstructural Proteins
  • Serine
  • Casein Kinase Ialpha
  • Protein Serine-Threonine Kinases
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • NS-5 protein, hepatitis C virus

Grants and funding

This work was supported by the National Health Research Institutes, Taiwan (http://english.nhri.org.tw/NHRI_WEB/nhriw001Action.do; NHRI-EX104-10213BI to MJY).