Epstein-Barr virus nuclear protein 2A forms oligomers in vitro and in vivo through a region required for B-cell transformation.
Epstein-Barr virus nuclear antigen 2 (EBNA-2) has been shown to be indispensable for immortalization of latently infected B lymphocytes, and it has been shown that EBNA-2 exists in a high-molecular-weight complex in these cells. In order to study the components of this protein machinery, we have purified baculovirus-expressed EBNA-2 from insect cells to greater than 95% homogeneity. We have shown by both gel filtration and sucrose gradient analysis that the purified material corresponds to a multimer containing eight EBNA-2 subunits. This multimeric complex is stable in 1.0 M NaCl, suggesting that the self-association is quite strong in vitro. By expressing portions of the EBNA-2 open reading frame to generate fusion proteins in yeast cells, we have used the two-hybrid system to demonstrate that this self-association occurs in vivo and is mediated at least in part by a domain of EBNA-2 encompassing amino acids 122 to 344. Mutational analysis of the self-association function suggests that two subdomains that flank amino acid 232 may each play a role in EBNA-2 protein-protein interaction.
Tsui
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Department of Medicine, State University of New York at Stony Brook.
Schubach
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D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001402:B-Lymphocytes / Q000166:cytology*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001704:Biopolymers; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019309:Epstein-Barr Virus Nuclear Antigens; D008969:Molecular Sequence Data; D009036:Moths; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000956:Antigens, Viral; D001704:Biopolymers; D017931:DNA Primers; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators