Purification and biochemical characterization of the Epstein-Barr virus-determined nuclear antigen and an associated protein with a 53,000-dalton subunit.
The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified 700-fold to apparent homogeneity from Raji and Namalwa cell extracts by a three-step procedure involving heat treatment, DNA-cellulose chromatography, and hydroxyapatite chromatography. Acid-fixed nuclear binding and complement fixation were used to monitor antigenic specificity. Purified EBNA was also capable of specifically inhibiting the regular anticomplement immunofluorescence reaction for EBNA against Raji target cells. The purified antigen had a molecular weight of 170,000 to 200,000. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it yielded a single 48,000-dalton (48K) monomer. An EBNA-associated protein was also purified from the same cell extract. It had a molecular weight of about 200,000 and yielded a single 53K protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same protein was also found in Epstein-Barr virus negative B-cell lymphoma lines. The two types of protein were characterized by amino acid composition and peptide mapping. The results showed that the 53K and 48K protein components have no long regions in common; this excludes that the smaller product arises by breakdown of the larger product. Residue distributions were different, but an excess of hydrophilic residues was found in both proteins, suggesting a certain overall similarity in properties. 53K components from different cell lines appeared to differ somewhat. Epstein-Barr virus-positive lines carry two 53K components, one of which may be a slightly modified 53K product. Immunocomplexing assay showed that the 48K, but not the 53K, protein carries EBNA specificity. In mixtures, the 53K protein is co-precipitated with the 48K protein. The data suggest that EBNA may form a complex with the 53K proten within the cell.
D000956:Antigens, Viral / Q000032:analysis / Q000302:isolation & purification*; D002460:Cell Line; D002467:Cell Nucleus / Q000276:immunology*; D000939:Epitopes; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D014764:Viral Proteins / Q000032:analysis / Q000302:isolation & purification*
D000956:Antigens, Viral; D000939:Epitopes; D010455:Peptides; D014764:Viral Proteins