Contribution of conserved amino acids in mediating the interaction between EBNA2 and CBF1/RBPJk.
The Epstein-Barr virus EBNA2 protein is a transcriptional activator that achieves promoter specificity through interaction with the cellular DNA-binding protein CBF1/RBPJk. Within the amino acid 252-to-425 EBNA2 domain that targets CBF1/RBPJk lie three amino acid clusters, conserved regions (CR) 5, 6, and 7, that are retained in the Epstein-Barr virus type A and type B and herpesvirus papio proteins. To further define the important features of the targeting domain, we constructed EBNA2 polypeptides containing deletions in the targeting domain and double or triple point mutations in the conserved motifs. The ability of these polypeptides and the type B and herpesvirus papio domains to interact 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 mutation of CR5 led to a moderate decrease in both activities. Mutation of CR7 had only a minor effect. Synthetic peptides corresponding to each of the conserved motifs were also used as competitors in an electrophoretic mobility shift assay. Only the peptide representing CR6 (amino acids 318 to 327), and not a version of this peptide mutated at the tryptophan residues at positions 323 and 324 (WW323,324), could compete for EBNA2 complex formation with CBF1/RBPJk. Overall, the data indicated that CR5 contributes to an optimal interaction, perhaps through stabilizing contacts, while CR6 forms a crucial interface with CBF1/RBPJk. The peptide competition data are consistent with direct contacts between WW323,324 and CBF1/RBPJk.
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
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Hayward
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000378:metabolism*; D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012995:Solubility; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation
D000956:Antigens, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D009687:Nuclear Proteins; D010455:Peptides