THE EPSTEIN-BARR-VIRUS IMMORTALIZING PROTEIN EBNA-2 IS TARGETED TO DNA BY A CELLULAR ENHANCER-BINDING PROTEIN

The Epstein-Barr virus nuclear antigen EBNA-2 is essential for Epstein -Barr virus-induced immortalization of B cells. EBNA-2 is a transcript ional activator capable of modifying the expression of specific viral and cellular genes. However, the mechanism of EBNA-2 transactivation h as been an enigma. We used a fractionated extract of CA46 lymphoblasto id cells and bacterially expressed EBNA-2 polypeptides to demonstrate that EBNA-2 is targeted to the Epstein-Barr virus latency C promoter ( Cp) through interaction with a cellular DNA binding protein designated Cp binding factor 1 (CBF1). A glutathione S-transferase-EBNA-2 fusion protein containing aa 252-425 of EBNA-2 interacted with CBF1 to yield a slowly migrating complex in an electrophoretic mobility shift assay . Mutation of EBNA-2 aa 323 and 324, which lie within a highly conserv ed amino acid motif, abolished the interaction with CBF1. This same mu tation also abolished the ability of EBNA-2 to activate the Cp in a co transfection assay. The binding site for CBF1 was localized to residue s -359 to -388 of the Cp by using an electrophoretic mobility shift as say and DNase I footprinting. Introduction of multiple copies of the C BF1 binding site upstream of a minimal heterologous promoter conferred EBNA-2 responsiveness on that promoter. Mutation of a core sequence C NGTGGGAA abolished CBF1 binding, and the mutated sequence was unable t o mediate EBNA-2 transactivation. The CBF1 core sequence also occurs i n other EBNA-2-responsive promoters suggesting that CBF1 may mediate E BNA-2 transactivation of both cellular and viral targets.