CONTRIBUTION OF CONSERVED AMINO-ACIDS IN MEDIATING THE INTERACTION BETWEEN EBNA2 AND CBF1 RBPJK/

The Epstein-Barr virus EBNA2 protein is a transcriptional activator th at achieves promoter specificity through interaction with the cellular DNA-binding protein CBF1/RBPJk. Within the amino acid 252-to-425 EBNA 2 domain that targets CBF1/RBPJk lie three amino acid clusters, conser ved regions (CR) 5, 6, and 7, that are retained in the Epstein-Barr vi rus type A and type B and herpesvirus papio proteins. To further defin e the important features of the targeting domain, we constructed E;BNA 2 polypeptides containing deletions in the targeting domain and double or triple point mutations in the conserved motifs. The ability of the se polypeptides and the type B and herpesvirus papio domains to intera ct with CBF1/RBPJk was examined by performing electrophoretic mobility shift assays and correlated with the effect of the mutations on EBNA2 transactivation. Both human type B EBNA2 and herpesvirus papio EBNA2 bound CBF1/RBPJk efficiently. Mutation of hydrophobic residues in CR6 severely impaired CBF1/RBPJk interaction and transactivation, while mu tation of CR5 led to a moderate decrease in both activities. Mutation of CR7 had only a minor effect. Synthetic peptides corresponding to ea ch of the conserved moths were also used as competitors in an electrop horetic mobility shift assay. Only the peptide representing CR6 (amino acids 318 to 327), and not a version of this peptide mutated at the t ryptophan residues at positions 323 and 324 (WW323,324), could compete for EBNA2 complex formation with CBF1/RBPJk. Overall, the data indica ted that CR5 contributes to an optimal interaction, perhaps through st abilizing contacts, while CR6 forms a crucial interface with CBF1/RBPJ k. The peptide competition data are consistent with direct contacts be tween WW323,324 and CBP1/RBPJk.