Characterization of the CBF2 binding site within the Epstein-Barr virus latency C promoter and its role in modulating...

The Epstein-Barr virus (EBV) EBNA2 protein is a transcriptional activator that regulates viral and cellular gene expression and is also essential for EBV-driven immortalization of B lymphocytes. The EBNA2-responsive enhancer in the viral latency C promoter (Cp) binds two cellular factors, CBF1 and CBF2. The precise role of the CBF2 protein for Cp enhancer function is presently unclear. CBF2 does not appear to interact with EBNA2 and binds just downstream of CBF1 between positions -339 and -368 in the Cp EBNA2 enhancer. Within this region an 8-bp sequence, CAGTGCGT, can be found, and a similar sequence is also located downstream of CBF1 binding sites in other EBNA2-responsive promoters. Previous studies have indicated that mutations and methylation in this sequence affect EBNA2 responsiveness. To investigate the requirements for CBF2 binding, we synthesized a series of oligonucleotides carrying double transversion mutations spanning both the conserved core sequence and outside flanking sequences. Surprisingly, mutations outside of the conserved core sequence in 4 bases immediately flanking the 5' end, GGTT, had the most deleterious effect on CBF2 binding. Mutations in the conserved core had a gradient effect, with those near the 5' end having the most deleterious effects on CBF2 binding. In addition, the affinities of CBF2 for binding to the LMP-1, LMP-2, and CD23 promoters were also measured. These promoters contain the conserved core but lack the 5' flanking GGTT motif and bound CBF2 weakly or not at all. Using Cp reporter plasmids containing CBF2 mutant binding sites, we were also able to show that at lower doses of EBNA2, Cp transactivation required a functional CBF2 binding site but that higher doses of EBNA2 transactivated CBF2 mutant promoters to 40% of wild-type levels. These assays indicate that CBF2 is important for EBNA2-mediated transactivation of the viral latency Cp. In addition, CBF2 activity was found to be associated with two polypeptides of 27 and 33 kDa.