Epstein-Barr virus (EBV) immortalizes resting human B cells very efficiently in vitro. The EBV nuclear protein EBNA2 is absolutely required for this process. It also activates transcription of cellular, as well as viral, genes. It is assumed that EBNA2 contributes to B cell immortalization by its transactivating potential, since its transforming and transactivating functions could not be separated. Mutational analysis of the 80 bp EBNA2 responsive cis-element within the viral bidirectional LMP/TP2 promoter region identified two sequence elements, which are both essential for transactivation by EBNA2. These sequences harbour putative consensus binding sites for Spi-1 oncoprotein and recombination signal binding protein RBP-J kappa, the homologue of Drosophila Suppressor of Hairless. Electrophoretic mobility shift assays demonstrated the high affinity binding of Spi-1 and Spi-B, both members of the Ets family of transcription factors, to one sequence element VSports手机版. The other element bound RBP-J kappa with low affinity. In addition, co-transfections showed that the replacement of the Spi-1/Spi-B binding site in the bi-directional LMP/TP2 promoter by the analogous SV40 Spi-1 responsive element did not impair its function on EBNA2-mediated transactivation. It is concluded that the transcriptional regulators Spi-1 and Spi-B as well as RBP-J kappa play an essential role in transactivating the LMP/TP2 promoter by EBNA2 and therefore in the immortalization of B cells by EBV. .