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We recently identified, by means of cotransformation of LTK- cells, a region of the Epstein-Barr virus (EBV) genome (the BamHI K fragment) that encodes or induces an EBV nuclear neoantigen (EBNA) serologically related to the EBNA found in lymphoid cells carrying the entire EBV genome. We now find that a second EBV DNA fragment, BamHI M, is also able to give rise to cotransformed LTK- cells with stable expression of a nuclear antigen. The BamHI K and M fragments have no apparent DNA homology. Many human sera that are reactive to EBNA in Raji cells detect both antigens; however, certain anti-EBNA-positive human sera are discordant and react only with the BamHI M or only with the BamHI K nuclear antigen. Every Raji cell appears to express both "M" and "K" antigens; D98 Raji cells, a somatic cell hybrid, express only "K" antigen. The K antigen is found on metaphase chromosomes of LTK cells and Raji cells. The M-induced antigen is not located on chromosomes when the cells are in metaphase but is present as granules within the nucleus.

A new antigenic polypeptide was identified in mouse L cells and in monkey COS-1 cells in which Epstein-Barr nuclear antigen (EBNA) was expressed as the result of gene transfer with cloned fragments of Epstein-Barr virus (EBV) DNA. The same-size protein (Mr, approximately equal to 78,000) was seen in stably transformed mouse cells harboring only the BamHI K fragment [approximately equal to 5.2 kilobase pairs (kbp)] or its BamHI/HindIII subfragment, I1f (approximately equal to 2.9 kbp). Thus, the latter DNA fragment is sufficient to code for the protein. In transfected COS cells, a deletion mutant of the I1f fragment (approximately equal to 2.3 kbp) gave rise to a truncated protein (Mr, approximately equal to 52,000), whereas the BamHI K fragment yielded a full-sized Mr 78,000 species. This finding indicates that EBNA is encoded by viral genes. In Burkitt lymphoma lines or in immortalized lymphocytes, variation in the size of the I1f fragment correlated with the apparent molecular weight of the EBNA polypeptide. EBNA is truncated in two Burkitt lymphoma lines, Raji (Mr, 67,000) and P3JHR-1 (Mr, 70,000), which have deletion mutant I1f genes. EBNA in human lymphoid cells bearing a complete I1f fragment as part of the entire EBV genome is the same size (Mr, 78,000) as EBNA found after gene transfer of I1f alone into mouse or monkey cells. Therefore, these expression systems make an authentic EBNA after transfer of the appropriate EBV genes.