Biochemical characterization of Epstein-Barr virus nuclear antigen 2A.
The Epstein-Barr virus nuclear antigen 2A (EBNA-2A) was immunoprecipitated from latently Epstein-Barr virus-infected lymphocytes with a polyclonal serum raised against the EBNA-2A C terminus. The nucleus contained three subfractions of EBNA-2A which could be distinguished by their resistance to salt extraction: (i) a nucleoplasmatic fraction that was solubilized at 50 mM NaCl, (ii) a chromatin-associated fraction extractable at 1.5 M NaCl, and (iii) a nuclear matrix-associated fraction solubilized only by boiling with buffer containing 2% sodium dodecyl sulfate. The three subfractions were phosphorylated; it was demonstrated that the nucleoplasmatic and the chromatin-associated fractions were phosphorylated at serine and threonine residues. The half-life of the EBNA-2A protein was determined by cycloheximide treatment and by pulse-chase experiments and was found to be at least 24 h. The turnover of the phosphate residues bound to the two salt-soluble subfractions was determined to be approximately 6 to 9 h, suggesting a possible role of the phosphorylation in the regulation of the biological activity of EBNA-2A. Dephosphorylation of EBNA-2A resulted in an increased mobility of the protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and indicated the presence of differentially phosphorylated subclasses of the protein. Analysis of EBNA-2A by sucrose gradient centrifugation revealed the existence of two subclasses of complexed molecules which exhibited sedimentation coefficients of approximately 13S and 34S.
Grässer
F A
FA
Abteilung Virologie, Universitätskliniken des Saarlandes, Homburg, Germany.
Haiss
P
P
Göttel
S
S
Mueller-Lantzsch
N
N
D000956:Antigens, Viral / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019309:Epstein-Barr Virus Nuclear Antigens; D004854:Herpesvirus 4, Human / Q000032:analysis / Q000378:metabolism*; D046911:Macromolecular Substances; D015394:Molecular Structure; D008970:Molecular Weight; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D011233:Precipitin Tests
D000956:Antigens, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D046911:Macromolecular Substances; D010750:Phosphoproteins