EPSTEIN-BARR NUCLEAR ANTIGEN-3 AND ANTIGEN-4 INTERACT WITH RBP-2N, A MAJOR ISOFORM OF RBP-J-KAPPA IN B-LYMPHOCYTES

The Epstein-Barr nuclear antigen (EBNA)-3 and EBNA-4 proteins are thou ght to act as transcriptional transactivators. The yeast two-hybrid sy stem and coimmunoprecipitation were used to demonstrate that EBNA-3 an d -4 associate with the DNA-binding protein RBP-2N, an isoform of RBP- 1 kappa. A comparison between EBNA-3, EBNA-4, and EBNA-6 binding to RB P-2N indicated that EBNA-3 enhanced beta-galactosidase activity 4-fold more than EBNA-6 and 30-fold more than EBNA-4. Assay of RBP-PN deleti on mutants demonstrated that EBNA-3 binds to regions of RBP-2N which a re distinct from those to which EBNA-2 and -6 interact, whereas EBNA-4 binds to the same region of RBP-2N as EBNA-2 and -6 (amino acids 159- 331 of RBP-2N). Interaction of both A- and B-type EBNA-3 with RBP-PN w as also demonstrated by immunoprecipitation. RT-PCR analysis of a pane l of B cell lymphomas and lymphoblastoid cell lines demonstrated that higher levels of RBP-PN were expressed, in comparison to RBP-J kappa, indicating that RBP-2N is a major isoform expressed in B cells. These results suggest that ail the EBNA-3 family proteins lead to transcript ional regulation via interaction with RBP-2N. (C) 1996 Academic Press, Inc.