EPSTEIN-BARR-VIRUS NUCLEAR-PROTEIN 2 TRANSACTIVATION OF THE LATENT MEMBRANE-PROTEIN-1 PROMOTER IS MEDIATED BY J-KAPPA AND PU1

Expression of the Epstein-Barr virus (EBV) latent membrane protein 1 ( LMP-1) oncogene is regulated by the EBV nuclear protein 2 (EBNA-2) tra nsactivator. EBNA-2 is known to interact with the cellular DNA-binding protein J kappa and is recruited to promoters containing the GTGGGAA J kappa recognition sequence. The minimal EBNA-2-responsive LMP-1 prom oter includes one J kappa-binding site, and we now show that mutation of that site, such that J kappa cannot bind, reduces EBNA-2 responsive ness by 60%. To identify other factors which interact with the LMP-1 E BNA-2 response element (EZRE), a -236/-145 minimal E2RE was used us a probe in an electrophoretic mobility shift assay. The previously chara cterized factors J kappa, PU.1, and AML1 bind to the LMP-1 E2RE, along with six other unidentified factors (LBF2 to LBF7). Binding sites wer e mapped for each factor. LBF4 is B- and T-cell specific and recognize s the PU.1 GGAA core sequence as shown by methylation interference. LB F4 has a molecular mass of 105 kDa and is probably unrelated to PU.1. LBF2 was found only in epithelial cell lines, whereas LBF3, LBF5, LBF6 , and LBF7 were not cell type specific. Mutations of the AML1- or LBF4 -binding sites had no effect on EBNA-2 transactivation, whereas mutati on of the PU.1-binding site completely eliminated EBNA-2 responses. A gst-EBNA-2 fusion protein specifically depleted PU.1 from nuclear extr acts and bound in vitro translated PU.1, providing biochemical evidenc e for a direct EBNA-ZPU.1 interaction. Thus, EBNA-2 transactivation of the LMP-1 promoter is dependent on interaction with at least two dist inct sequence-specific DNA-binding proteins, J kappa and PU.1. LBF3, L BF5, LBF6, or LBF7 may also be involved, since their binding sites als o contribute to EBNA-2 responsiveness.