Purification of a polypeptide complex (p52) belonging to the D-subspecificites of Epstein-Barr virus-induced early antigens

A two-dimensional immunoblot analysis of chemically induced EBV DNA carrying Burkitt's lymphoma cell lines shows besides a large number of minor components at least two major groups of polypeptides: the most prominent group of polypeptides is observed in the range of 48 to 58 kDa (p I 4.5 to 8.5) and another group at 38 36 kDa (p I 4.4). A polypeptide complex (p52) belonging to the Epstein-Barr virus (EBV)-induced early antigen complex (EA) has been isolated from IdU-induced Raji and B95-8 cells as well as from n-butyrate-induced P3HR-1 cells. The p52 polypeptides have been purified by chromatography on Blue-, DEAE-, CM-, and Phenyl-Sepharose. The purification of these components of the EA complex was monitored by ELISA and by two-dimensional immunoblots using a well-characterized high anti-EBV positive human serum. The isolated polypeptides have an apparent mol wt of about 52,000 Da as determined under nondenaturing conditions by gel filtration chromatography on Sephacryl S-300. One- and two-dimensional immunoblots show a major group of polypeptides of 52 kDa (p I 8.5 to 5.5) with EA activity and some minor components with smaller size up to 40 kDa. The latter seem to be generated by limited proteolysis of p52 polypeptides. The EA activity of the isolated polypeptides could be confirmed by their reaction with IgG anti-EA positive as well as IgA anti-EA positive sera by ELISA. The purified polypeptide complex did not react with anti-EA-D negative, anti-EA-R positive sera obtained from patients with African Burkitt's lymphoma, suggesting that these polypeptides belong to the EA-D complex. The monoclonal antibody R3 reacted with the isolated 52 kDa components of EA suggesting a common epitope present on these polypeptides, the same result was obtained with three rabbit sera produced against the isolated polypeptide complex.