Discordant expression of 2 Epstein-Barr virus-associated antigens, EBNA and RANA, in man-rodent somatic cell hybrids

The Epstein Barr nuclear antigen (EBNA) and the rheumatoid arthritis nuclear antigen (RANA) develop in human B lymphocytes that have been infected and transformed by Epstein Barr virus (EBV). Antibodies to RANA and EBNA are found only in individuals with prior exposure to EBV. The purpose of the present studies was to determine the relation of the 2 antigens to each other and to EBV genetic material, in human-rodent somatic cell hybrids. Cultured human B lymphoblastoid cells, Raji, Daudi, and RPMI 4098 were fused with thymidine kinase-deficient mouse or hamster fibroblasts. After selection and cloning in ouabain-HAT medium, the hybrid nature of the surviving cells was confirmed by isozyme analysis. The hybrid clones were analyzed for EBNA by anti-complement im,munofluorescence, and for RANA by anti-immunoglobulin immunofluorescence and immunodiffusion. The results showed that RANA and EBNA segregated entirely independently of each other in the hybrid clones. Two methods were used to detect the presence of EBV DNA sequences in the intracellular DNA of hybrid clones. The 1st method relied on the hybridization of labeled cRNA prepared from virion DNA with DNA from 8 hybrid clones affixed to nitrocellulose filters. The 2nd approach was to hybridize labeled intracellular DNA from 3 hybrid clones to Southern blots of cloned fragments of EBV DNA. These results suggested that the presence of EBV DNA was not sufficient for the expression of either antigen. One stable RANA-positive hybrid cell line contained at least 80% of the EBV genome in the absence of detectable EBNA.