Characterization of the EBV BGLF4 Induced Phosphorylation of EA-D and Its Effect on EA-D Function

• Main Authors: YI-HSIN CHOU, 周以欣
• Other Authors: 陳美如
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/19832689401860214062

碩士 === 國立臺灣大學 === 微生物學研究所 === 90 === The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a Ser/ Thr protein kinase based on the homology alignment to the conserved motifs of known protein kinases. According to the EBV genome map, BGLF4 is located at nucleotides (nt) 123,692-122,328 of B95-8 EBV. Since no in frame ATG was identified at nt 123,692, the first in frame ATG at nt 123,614 was used to express the BGLF4 protein in our previous studies. By using an EBNA-1 tag, the immunoprecipitated BGLF4 was demonstrated for its abilities of autophosphorylation and phosphorylating casein, histone, and EBV early-antigen diffuse type (EA-D). In order to understand whether BGLF4 phosphorylates EA-D in vivo, plasmids expressing BGLF4ATG and EA-D were cotransfected into 293T cells in this study. Molecular weight shift of EA-D was observed in cotransfected cells, and this modification was removed by calf intestinal alkaline phosphatase (CIP) treatment in vitro. In addition, the phosphorylation of EA-D down regulated its transactivation function on BHLF1 promoter in a luciferase reporter system. We also demonstrated that both BGLF4ATG and BGLG4-78, which including upstream 78 base pair, could phosphorylate EA-D and down regulate its transactivation function in a similar pattern. BHLF1 gene is the most actively transcribed gene during early EBV replication, although the precise function of the BHLF1 gene product remains unknown. However, since BHLF1 gene locates next to EBV oriLyt, it was suggested that BHLF1 may be involved in EBV DNA replication. Further investigation of the biological significance of BGLF4 regulation on EA-D will be important for understanding possible cascade control of EBV gene expression in lytic cycle.