https://scholars.lib.ntu.edu.tw/handle/123456789/508014
標題: | Glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus mucleocapsid protein and viral replication | 作者: | Wu C.-H. Shiou-Hwei Yeh Tsay Y.-G. Shieh Y.-H. CHUAN-LIANG KAO Chen Y.-S. Wang S.-H. Kuo T.-J. Chen D.-S. PEI-JER CHEN |
公開日期: | 2009 | 卷: | 284 | 期: | 8 | 起(迄)頁: | 5229-5239 | 來源出版物: | Journal of Biological Chemistry | 摘要: | Coronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication. ? 2009 by The American Society for Biochemistry and Molecular Biology, Inc. |
URI: | https://scholars.lib.ntu.edu.tw/handle/123456789/508014 | ISSN: | 0021-9258 | DOI: | 10.1074/jbc.M805747200 | SDG/關鍵字: | Amino acids; Enzymes; Life cycle; Consensus sequence; Constitutively actives; Coronavirus; Cytopathic effect; Deletion mapping; Glycogen synthase kinase; Glycogen synthase kinase-3; Hepatitis virus; High resolution; Immunoprecipitation; In-vitro; Isoforms; Mass spectrometric analysis; Nucleocapsid; Page analysis; Phosphorylated proteins; Phosphorylation sites; Protein phosphorylation; Severe acute respiratory syndrome coronavirus; Severe acute respiratory syndrome CoV; Specific inhibitors; Viral life cycle; Viral replication; Phosphorylation; arginine; glycogen synthase kinase 3; glycogen synthase kinase 3 inhibitor; nucleocapsid protein; serine; glycogen synthase kinase; nucleocapsid protein; nucleocapsid protein, Coronavirus; protein kinase inhibitor; amino acid sequence; article; controlled study; cytopathogenic effect; enzyme regulation; gene deletion; gene mapping; human; human cell; immunoprecipitation; mass spectrometry; nonhuman; nucleotide sequence; polyacrylamide gel electrophoresis; priority journal; protein dephosphorylation; protein motif; protein phosphorylation; SARS coronavirus; severe acute respiratory syndrome; Vero cell; virus replication; virus strain; animal; Cercopithecus; consensus sequence; drug effect; genetics; metabolism; methodology; mouse; Murine hepatitis coronavirus; mutation; peptide mapping; phosphorylation; physiology; SARS coronavirus; Coronavirus; Murine hepatitis virus; SARS coronavirus; Amino Acid Motifs; Animals; Cercopithecus aethiops; Consensus Sequence; Cytopathogenic Effect, Viral; Glycogen Synthase Kinases; Humans; Mice; Murine hepatitis virus; Mutation; Nucleocapsid Proteins; Peptide Mapping; Phosphorylation; Protein Kinase Inhibitors; SARS Virus; Vero Cells; Virus Replication |
顯示於: | 醫學檢驗暨生物技術學系 |
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