title: Phosphorylation of Serine 235 of the Hepatitis C Virus Non-Structural Protein NS5A by Multiple Kinases.
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.
authors.affiliation: Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan.
authors.forename: Kuan-Ying
authors.initials: KY
authors.lastname: Lee
authors.affiliation: Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan.
authors.forename: Yi-Hung
authors.initials: YH
authors.lastname: Chen
authors.affiliation: Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan.
authors.forename: Shih-Chin
authors.initials: SC
authors.lastname: Hsu
authors.affiliation: Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan.
authors.forename: Ming-Jiun
authors.initials: MJ
authors.lastname: Yu
mesh_terms: D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism*; D048128:Casein Kinase Ialpha / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D055785:Gene Knockdown Techniques; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*; D000097869:Polo-Like Kinase 1; D012324:RNA-Dependent RNA Polymerase
chemical_list: D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048128:Casein Kinase Ialpha; D018797:Cell Cycle Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D012694:Serine; D017361:Viral Nonstructural Proteins; D000097869:Polo-Like Kinase 1; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase