Epstein–Barr Nuclear Antigen-3 and -4 Interact with RBP-2N, a Major Isoform of RBP-Jκ in B Lymphocytes

• Published in: Virology (New York, N.Y.) N.Y.), 1996-12, Vol.226 (2), p.346-353
• Main Authors: Krauer, Kenia G., Kienzle, Norbert, Young, David B., Sculley, Tom B.
• Format: Article
• Language: English
• Subjects: B-Lymphocytes - metabolism; B-Lymphocytes - virology; beta-Galactosidase - metabolism; Cell Line; Enzyme Activation; Epstein-Barr Virus Nuclear Antigens - metabolism; Herpesvirus 4, Human - metabolism; Humans; Precipitin Tests; Protein Binding; Repressor Proteins - metabolism; Saccharomyces cerevisiae - genetics; Sequence Deletion; Tumor Cells, Cultured
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1006/viro.1996.0662

The Epstein–Barr nuclear antigen (EBNA)-3 and EBNA-4 proteins are thought to act as transcriptional transactivators. The yeast two-hybrid system and coimmunoprecipitation were used to demonstrate that EBNA-3 and -4 associate with the DNA-binding protein RBP-2N, an isoform of RBP-Jκ. A comparison between EBNA-3, EBNA-4, and EBNA-6 binding to RBP-2N indicated that EBNA-3 enhanced β-galactosidase activity 4-fold more than EBNA-6 and 30-fold more than EBNA-4. Assay of RBP-2N deletion mutants demonstrated that EBNA-3 binds to regions of RBP-2N which are 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-2N was also demonstrated by immunoprecipitation. RT-PCR analysis of a panel of B cell lymphomas and lymphoblastoid cell lines demonstrated that higher levels of RBP-2N were expressed, in comparison to RBP-Jκ, indicating that RBP-2N is a major isoform expressed in B cells. These results suggest that all the EBNA-3 family proteins lead to transcriptional regulation via interaction with RBP-2N.