https://scholars.lib.ntu.edu.tw/handle/123456789/452849
標題: | Serine 229 balances the hepatitis C virus nonstructural protein NS5A between hypo- And hyperphosphorylated states | 作者: | Tsai C.-N. Pan T.-C. Chiang C.-H. Yu C.-C. Su S.-H. MING-JIUN YU |
關鍵字: | Hepatitis C virus; Protein kinases; Protein phosphorylation; Proteomics; Viral replication | 公開日期: | 2019 | 出版社: | American Society for Microbiology | 卷: | 9 | 期: | 23 | 來源出版物: | Journal of Virology | 摘要: | The nonstructural protein NS5A of hepatitis C virus (HCV) is a phosphorylated protein that is indispensable for viral replication and assembly. We previously showed that NS5A undergoes sequential serine S232/S235/S238 phosphorylation resulting in NS5A transition from a hypo- to a hyperphosphorylated state. Here, we studied functions of S229 with a newly generated antibody specific to S229 phosphorylation. In contrast to S232, S235, or S238 phosphorylation detected only in the hyperphosphorylated NS5A, S229 phosphorylation was found in both hypo- and hyperphosphorylated NS5A, suggesting that S229 phosphorylation initiates NS5A sequential phosphorylation. Immunoblotting showed an inverse relationship between S229 phosphorylation and S235 phosphorylation. When S235 was phosphorylated as in the wild-type NS5A, the S229 phosphorylation level was low; when S235 could not be phosphorylated as in the S235A mutant NS5A, the S229 phosphorylation level was high. These results suggest an intrinsic feedback regulation between S229 phosphorylation and S235 phosphorylation. It has been known that NS5A distributes in large static and small dynamic intracellular structures and that both structures are required for the HCV life cycle. We found that S229A or S229D mutation was lethal to the virus and that both increased NS5A in large intracellular structures. Similarly, the lethal S235A mutation also increased NS5A in large structures. Likewise, the replication-compromised S235D mutation also increased NS5A in large structures, albeit to a lesser extent. Our data suggest that S229 probably cycles through phosphorylation and dephosphorylation to maintain a delicate balance of NS5A between hypo- and hyperphosphorylated states and the intracellular distribution necessary for the HCV life cycle. IMPORTANCE This study joins our previous efforts to elucidate how NS5A transits between hypo- and hyperphosphorylated states via phosphorylation on a series of highly conserved serine residues. Of the serine residues, serine 229 is the most interesting since phosphorylation-mimicking and phosphorylation-ablating mutations at this serine residue are both lethal. With a new high-quality antibody specific to serine 229 phosphorylation, we concluded that serine 229 must remain wild type so that it can dynamically cycle through phosphorylation and dephosphorylation that govern NS5A between hypo- and hyperphosphorylated states. Both are required for the HCV life cycle. When phosphorylated, serine 229 signals phosphorylation on serine 232 and 235 in a sequential manner, leading NS5A to the hyperphosphorylated state. As serine 235 phosphorylation is reached, serine 229 is dephosphorylated, stopping signal for hyperphosphorylation. This balances NS5A between two phosphorylation states and in intracellular structures that warrant a productive HCV life cycle. Copyright ? 2019 American Society for Microbiology. All Rights Reserved. |
URI: | https://www.scopus.com/inward/record.uri?eid=2-s2.0-85074965586&doi=10.1128%2fJVI.01028-19&partnerID=40&md5=c6c0e8b8e6f4b8bf24b75625520cd56f https://scholars.lib.ntu.edu.tw/handle/123456789/452849 |
ISSN: | 0022538X | DOI: | 10.1128/JVI.01028-19 | SDG/關鍵字: | nonstructural protein 5A; serine; NS-5 protein, hepatitis C virus; protein kinase; serine; viral protein; amino acid sequence; Article; cell structure; cellular distribution; controlled study; feedback system; Hepatitis C virus; human; human cell; immunoblotting; life cycle; mutant; mutation; nonhuman; priority journal; protein dephosphorylation; protein phosphorylation; virus replication; wild type; gene mutation; HEK293-A cell line; Huh-7.5.1 cell line; protein localization; virogenesis; virus assembly; animal; chemistry; genetics; HEK293 cell line; Hepacivirus; hepatitis C; metabolism; phosphorylation; physiology; proteomics; Renilla; tumor cell line; virology; Amino Acid Sequence; Animals; Cell Line, Tumor; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Phosphorylation; Protein Kinases; Proteomics; Renilla; Serine; Viral Nonstructural Proteins; Virus Replication |
顯示於: | 生物化學暨分子生物學科研究所 |
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