Monoclonal and polyclonal antibodies against Epstein-Barr virus nuclear antigen 5 (EBNA-5) detect multiple protein species in Burkitt's lymphoma and lymphoblastoid cell lines

Abstract

The Epstein-Barr virus nuclear antigen 5 (EBNA-5) is encoded by highly spliced mRNA from the major IR1 (BamHI-W) repeat region of the virus genome. A mouse monoclonal antibody, JF186, has been raised against a synthetic 18-amino-acid peptide deduced from the EBNA-5 message of B95-8 and Raji cells. The antibody showed characteristic coarse nuclear granules by indirect immunofluorescence and revealed multiple EBNA-5 species by immunoblotting and immunoprecipitation. The B95-8 line itself and all B95-8 virus-carrying cells, whether lymphoblastoid cell lines or in vitro-converted sublines of Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) lines, were EBNA-5 positive. Among 36 cell lines carrying different EBV strains, only 10 expressed the B95-8-Raji-prototype EBNA-5 recognized by JF186; this was probably due to genetic variation in the epitope recognized by JF186, as shown for P3HR-1. Human antibodies, affinity purified against EBNA-5-JF186 immunoprecipitates, detected EBNA-5 in the majority of EBV-positive BL lines and in all lymphoblastoid cell lines containing the BL-derived viruses. Thus, EBNA-5 can be expressed by all virus isolates examined, but is down-regulated, together with other latent gene products, in a minority of BL lines which have a particular cellular phenotype. EBNA-5 was detected as a ladder of protein species of 20 to 130 kilodaltons (kDa), with a regular spacing of 6 to 8 kDa, consistent with the coding capacity of the combined BamHI-W 66- and 132-base-pair exons, together with shifts of 2 to 4 kDa, consistent with the size of the separate 66- and 132-base-pair exons. Multiple EBNA-5 proteins can be expressed by the single cell as shown by cloning of newly infected cells.