EBNA-2 UP-REGULATION OF EPSTEIN-BARR-VIRUS LATENCY PROMOTERS AND THE CELLULAR CD23 PROMOTER UTILIZES A COMMON TARGETING INTERMEDIATE, CBF1

The EBNA-2 protein is essential for the establishment of a latent Epst ein-Barr virus (EBV) infection and for B-cell immortalization. EBNA-2 functions as a transcriptional activator that modulates viral latency gene expression as well as the expression of cellular genes, including CD23. We recently demonstrated that EBNA-2 transactivation of the EBV latency C promoter (Cp) is dependent on an interaction with a cellula r DNA-binding protein, CBF1, for promoter targeting. To determine whet her targeting via CBF1 is a common mechanism for EBNA-2-mediated trans activation, we have examined the requirements for activation of the ce llular CD23 promoter. Binding of CBF1 to a 192-bp mapped EBNA-2-respon sive region located at position -85 bp to -277 bp upstream of the CD23 promoter was detected in electrophoretic mobility shift assays. The i dentity of the bound protein as CBF1 was established by showing that t he bound complex was competed for by the CBF1 binding site from the EB V Cp, that the bound protein could be supershifted with a bacterially. expressed fusion protein containing amino acids 252 to 425 of EBNA-2 but was unable to interact with a non-CBF1-binding EBNA-2 mutant (WW32 3SR), and that in W cross-linking experiments, the Cp CBF1 binding sit e and the CD23 probe bound proteins of the same size. The requirement for interaction with CBF1 was demonstrated in a transient cotransfecti on assay in which the multimerized 192-bp CD23 response region was tra nsactivated by wild-type EBNA-2 but not by the WW323SR mutant. Reporte r constructions carrying multimerized copies of the 192-bp CD23 respon se region or multimers of the CBF1 binding site from the CD23 promoter were significantly less responsive to EBNA-2 transactivation than equ ivalent constructions carrying a multimerized region from the Cp or mu ltimers of the CBF1 binding site from the Cp. Direct binding and compe tition assays using 30-mer oligonucleotide probes representing the ind ividual CBF1 binding sites indicated that CBF1 bound less efficiently to the CD23 promoter and the EBV LMP-1 promoter sites than to the Cp s ite. To investigate the basis for this difference, we synthesized a se ries of oligonucleotides carrying mutations across the CBF1 binding si te and used these as competitors in electrophoretic mobility shift ass ays. The competition experiments indicated that a central core sequenc e, GTGGGAA, common to all known EBNA-2-responsive elements, is crucial for CBF1 binding. Flanking sequences on either side of this core infl uence the affinity for CBF1. The Cp has the most optimal binding site, with the CD23 site having an intermediate affinity and the LMP-1 site having a low affinity for CBF1. These differences in affinity can be directly correlated with changes in the flanking sequences. A search o f databases using the CBF1 site defined in this study indicates that t he number of cellular genes which can potentially be subject to EBNA-2 activation may be considerably larger than previously appreciated.