Characterization of the Epstein-Barr virus-induced early polypeptide complex p50/58 EA-D using rabbit antisera, a...

A polypeptide complex (p52) belonging to the D-subspecificity of the EBV-induced early antigens (EA-D) was purified from chemically induced P3HR-1 cells. Rabbit antisera raised against the isolated polypeptides reacted with components of EA-D as could be shown by indirect immunofluorescence and immunoperoxidase staining of IdU-induced EA positive Raji cells, ELISA, and immunoblotting. In one-dimensional immunoblots the rabbit antisera detected a predominant polypeptide complex of 52 kDa. Two-dimensional immunoblots prepared with proteins from IdU-induced Raji cells showed that the rabbit sera detect three series of polypeptides of 52 kDa (pl 8.5-6.2), 55-58 kDa (pl 6.2-4.5), and 48-50 kDa (pl 6.0-4.5). These three groups of polypeptides could also be identified by 50 high titered anti-EA-D positive human sera and a specific monoclonal antibody (R3) as being the main components of EA-D in Raji and B95-8 cells. All polypeptides of the p50/58 complex showed DNA binding properties either by themselves or by an interaction with other proteins. When TPA or IdU-induced Raji cells were labeled with 2Pi, two phosphorylated polypeptides pp50 and pp58 could be immunoprecipitated with the rabbit sera and a high anti-EA titered human serum. The time course of the synthesis of polypeptides associated with the EA-D complex was studied by 2-D immunoblots: EA polypeptides of 52 kDa appeared as early as 6 hr after the addition of IdU to Raji cells in culture, polypeptides of 55-58 and 48-50 kDa after 18 and 25 hr, respectively. The coordinated appearance of these groups of polypeptides and their similar size and reactivity with human sera and rabbit antisera produced against the isolated p52 as well as with a monoclonal antibody (R3) suggested that most of these polypeptides are derived from post-translational modifications of one or a few initially synthesized polypeptides, possibly p52. Phosphorylation seems to be at least one possibility of post-translational modification.