https://scholars.lib.ntu.edu.tw/handle/123456789/288752
標題: | A protein kinase activity associated with Epstein-Barr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro | 作者: | MEI-RU CHEN Chang, Shin-Jye Huang, Hsiaowen Chen, Jen-Yang |
公開日期: | 2000 | 卷: | 74 | 期: | 7 | 起(迄)頁: | 3093-3104 | 來源出版物: | Journal of Virology | 摘要: | The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a potential Ser/Thr protein kinase gene through the recognition of amino acid sequence motifs characteristic of conserved regions within the catalytic domains of protein kinases. In order to investigate this potential kinase activity, BGLF4 was expressed in Escherichia coli and the purified protein was used to generate a specific antiserum. Recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase, was used to infect 293 and 293T cells after transient transfection with a plasmid containing BGLF4 under the control of the T7 promoter. Autophosphorylation of the BGLF4 protein was demonstrated using the specific antiserum in an immune complex kinase assay. In addition, EBNA-1-tagged BGLF4 and EBNA-1 monoclonal antibody 5C11 were used to demonstrate the specificity of the kinase activity and to locate BGLF4 in the cytoplasm of transfected cells. Manganese ions were found to be essential for autophosphorylation of BGLF4, and magnesium can stimulate the activity. BGLF4 can utilize GTP, in addition to ATP, as a phosphate donor in this assay. BGLF4 can phosphorylate histone and casein in vitro. Among the potential vital protein substrates we examined, the EBV early antigen (EA-D, BMRF1), a DNA polymerase accessory factor and an important transactivator during lytic infection, was found to be phosphorylated by BGLF4 in vitro. Amino acids 1 to 26 of BGLF4, but not the predicted conserved catalytic domain, were found to be essential for autophosphorylation of BGLF4. |
URI: | http://www.scopus.com/inward/record.url?eid=2-s2.0-0034011617&partnerID=MN8TOARS http://scholars.lib.ntu.edu.tw/handle/123456789/288752 |
DOI: | 10.1128/JVI.74.7.3093-3104.2000 | SDG/關鍵字: | early antigen; protein kinase; virus antigen; article; autophosphorylation; catalysis; disease association; enzyme activity; enzyme phosphorylation; Epstein Barr virus; genetic transfection; in vitro study; nonhuman; priority journal; protein domain; transactivation; virus cell interaction; virus recombination; Amino Acid Sequence; Antigens, Viral; Cloning, Molecular; Cytoplasm; Enzyme Inhibitors; Escherichia coli; Heparin; Herpesvirus 4, Human; Immune Sera; Kinetics; Molecular Sequence Data; Okadaic Acid; Phosphorylation; Protein Kinases; Protein-Serine-Threonine Kinases; Sequence Homology, Amino Acid; Serine; Threonine; Viral Proteins |
顯示於: | 醫學系 |
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