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We used antiserum raised against the bacterially synthesized product of one of the open reading frames in Epstein-Barr virus (EBV) BamHI fragment M to demonstrate that this reading frame (BMRF1) codes for a nuclear protein of the diffuse early antigen (EA) class. In indirect immunofluorescence assays, the rabbit anti-BMRF1 antiserum gave nuclear staining in approximately 5% of Raji cells which had been treated with sodium butyrate, and positive fluorescence was observed in both acetone- and methanol-fixed cells. Uninduced Raji cultures contained less than 0.1% positive cells regardless of whether indirect immunofluorescence or anti-complement immunofluorescence was used. In immunoblot analyses, the rabbit serum identified a family of polypeptides of 46 to 55 kilodaltons (kDa) in total protein extracts from B95-8 cells or from butyrate-induced Raji cells. In both cell types, the dominant polypeptides were the 48- and 50-kDa species. This same family of polypeptides was identified when the immunoblots were reacted with the R3 monoclonal antibody, and we concluded that this antibody also recognized the product of the BMRF1 open reading frame. Fibroblast cell lines containing EBV BamHI fragment M were established by cotransfection of baby hamster kidney cells with BamHI-M and the gene for neomycin resistance. Aminoglycoside G418-resistant colonies which showed evidence for EBV antigen expression in immunofluorescence assays were selected, and clonal cell lines were established. After 3 to 4 months of passaging, constitutive synthesis of EA was no longer detectable in these cell lines either by immunofluorescence or by immunoblot analysis. However, in the one cell line examined, synthesis of the 48- to 50-kDa EA was induced by treatment of the culture with sodium butyrate. Thus, the regulation of expression of this EA in transfected fibroblasts is analogous to that seen in Raji lymphoblasts. We showed previously that BamHI fragment M also contains the coding sequences for a 60-kDa nuclear EA, and hence BamHI-M encodes two separate components of the diffuse EA complex.

A second Epstein-Barr virus early antigen gene in BamHI fragment M encodes a 48- to 50-kilodalton nuclear protein