title: Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.
abstract: The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein.
authors.affiliation: From the Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Rm. 816, No. 1 Sec. 1 Jen-Ai Road, Taipei 10051, Taiwan and.
authors.forename: Weng Man
authors.initials: WM
authors.lastname: Chong
authors.affiliation: From the Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Rm. 816, No. 1 Sec. 1 Jen-Ai Road, Taipei 10051, Taiwan and.
authors.forename: Shih-Chin
authors.initials: SC
authors.lastname: Hsu
authors.affiliation: From the Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Rm. 816, No. 1 Sec. 1 Jen-Ai Road, Taipei 10051, Taiwan and.
authors.forename: Wei-Ting
authors.initials: WT
authors.lastname: Kao
authors.affiliation: From the Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Rm. 816, No. 1 Sec. 1 Jen-Ai Road, Taipei 10051, Taiwan and.
authors.forename: Chieh-Wen
authors.initials: CW
authors.lastname: Lo
authors.affiliation: From the Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Rm. 816, No. 1 Sec. 1 Jen-Ai Road, Taipei 10051, Taiwan and.
authors.forename: Kuan-Ying
authors.initials: KY
authors.lastname: Lee
authors.affiliation: From the Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Rm. 816, No. 1 Sec. 1 Jen-Ai Road, Taipei 10051, Taiwan and.
authors.forename: Jheng-Syuan
authors.initials: JS
authors.lastname: Shao
authors.affiliation: From the Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Rm. 816, No. 1 Sec. 1 Jen-Ai Road, Taipei 10051, Taiwan and.
authors.forename: Yi-Hung
authors.initials: YH
authors.lastname: Chen
authors.affiliation: From the Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Rm. 816, No. 1 Sec. 1 Jen-Ai Road, Taipei 10051, Taiwan and.
authors.forename: Justin
authors.initials: J
authors.lastname: Chang
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.
authors.forename: Steve S-L
authors.initials: SS
authors.lastname: Chen
authors.affiliation: From the Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Rm. 816, No. 1 Sec. 1 Jen-Ai Road, Taipei 10051, Taiwan and mjyu@ntu.edu.tw.
authors.forename: Ming-Jiun
authors.initials: MJ
authors.lastname: Yu
mesh_terms: D047389:Casein Kinase I / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation; D040901:Proteomics; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D047389:Casein Kinase I; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: Hepatitis C virus (HCV); antibody; casein kinase; phosphoproteomics; phosphorylation; proteomics; serine 235