Accumulation of Epstein-Barr virus (EBV) BMRF1 protein EA-D during latent EBV activation of Burkitt's lymphoma cell line Raji.
As a new model to elucidate molecular mechanisms in Epstein-Barr virus (EBV) activation, we tested the tetracycline-inducible (Tet-On)/BZLF1-oriP plasmid system in Raji cells. Cells transfected with this Tet-On plasmid did not activate EBV by doxycycline and surprisingly EBV latency was disrupted with large amounts of BMRF1 protein (EA-D) being accumulated in the cells. Brilliant EA-D fluorescence was markedly condensed in small sized cells, intra-cellular vesicles, and extra-cellular particles. Scanning electron microscopy demonstrated the extra-cellular particles to be covered with a membrane. EA-D molecules of 58, 50, 48, and 44kDa were expressed in the cells. The high (58 and 50kDa) and low (48 and 44kDa) EA-D molecules appeared in the early and late stages, respectively. Low EA-D molecules were detected mostly in EA-D positive cells separated into the heaviest density layer of a discontinuous Percoll gradient. Such molecules could be created from high EA-D molecules by protein phosphatase treatment. The EA-D molecules that appeared similar were detected in EBV-activated P3HR-1 and Akata cells. Several hypotheses concerning the accumulation of EA-D molecules of various polymorphic forms and their phosphorylation/dephosphorylation in this model system are presented, with possible biological and clinical relevance.
Ohashi
Makoto
M
Division of Biosignaling, Department of Biomedical Science, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago 683-8503, Japan.
Horie
Kazutaka
K
Hoshikawa
Yoshiko
Y
Nagata
Keiko
K
Osaki
Mistuhiko
M
Ito
Hisao
H
Sairenji
Takeshi
T
D000956:Antigens, Viral / Q000096:biosynthesis*; D015153:Blotting, Western; D002051:Burkitt Lymphoma / Q000737:chemistry / Q000821:virology*; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D003593:Cytoplasm / Q000737:chemistry; D022162:Cytoplasmic Vesicles / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D004854:Herpesvirus 4, Human / Q000254:growth & development*; D006801:Humans; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D013752:Tetracycline; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D014775:Virus Activation
D000956:Antigens, Viral; C063676:BZLF1 protein, Herpesvirus 4, Human; D004268:DNA-Binding Proteins; C083204:Epstein-Barr virus early antigen diffuse component; D015534:Trans-Activators; D014764:Viral Proteins; D010749:Phosphoprotein Phosphatases; D013752:Tetracycline