Detection of Epstein-Barr virus BGLF4 protein kinase in virus replication compartments and virus particles.
BGLF4 is the only serine/threonine protein kinase identified in Epstein-Barr virus (EBV); it is known to phosphorylate viral DNA polymerase processivity factor, EA-D (BMRF1), EBNA-LP, EBNA-2, cellular EF-1delta and nucleoside analogue ganciclovir. However, the expression and biological functions of BGLF4 have not yet been clearly demonstrated in EBV-infected cells. To reveal authentic functions of BGLF4 protein within viral-replicating cells, a panel of specific monoclonal antibodies was generated and characterized. The major immunogenic regions of BGLF4 were mapped to aa 27-70 and 327-429. Using these antibodies, the expression kinetics and localization of BGLF4 were analysed in reactivated EBV-positive lymphoid and epithelial cells. BGLF4 was expressed as a phosphoprotein at the early lytic stage and was detected predominantly in the nucleus of EBV-positive cells, but small amounts of BGLF4 were observed in cytosolic and heavy membrane fractions at the late phase of virus replication. Additionally, it was demonstrated that BGLF4 co-localizes with viral DNA polymerase processivity factor, EA-D (BMRF1), in the virus replication compartment and that it is a virion component. Finally, possible functional domains at the N terminus of BGLF4 were analysed and it was found that aa 1-26 of BGLF4 are dispensable for EA-D phosphorylation, whereas deletion of aa 27-70 reduced kinase activity.
Wang
Jiin-Tarng
JT
Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, No. 1 1st Section Jen-Ai Road, Taipei, Taiwan.
Yang
Pei-Wen
PW
Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, No. 1 1st Section Jen-Ai Road, Taipei, Taiwan.
Lee
Chung-Pei
CP
Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, No. 1 1st Section Jen-Ai Road, Taipei, Taiwan.
Han
Chia-Hong
CH
Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, No. 1 1st Section Jen-Ai Road, Taipei, Taiwan.
Tsai
Ching-Hwa
CH
Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, No. 1 1st Section Jen-Ai Road, Taipei, Taiwan.
Chen
Mei-Ru
MR
Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, No. 1 1st Section Jen-Ai Road, Taipei, Taiwan.
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000032:analysis / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004847:Epithelial Cells / Q000737:chemistry / Q000821:virology; D015870:Gene Expression; D004854:Herpesvirus 4, Human / Q000737:chemistry*; D008214:Lymphocytes / Q000737:chemistry / Q000821:virology; D051379:Mice; D010766:Phosphorylation; D010770:Phosphotransferases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D017384:Sequence Deletion; D013194:Staining and Labeling; D013997:Time Factors; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; C083204:Epstein-Barr virus early antigen diffuse component; D014764:Viral Proteins; D010770:Phosphotransferases; C406063:BGLF4 protein, Epstein-Barr virus; D017346:Protein Serine-Threonine Kinases