The Epstein-Barr virus (EBV) DNA polymerase (pol) is essential for the replication of viral genomes during productive EBV infection. We have previously reported that the EBV DNA pol promoter, which is TATA-less and constitutively inactive, is activated by a genomic clone expressing both immediate-early viral transactivators, BZLF1Z and BRLF1 (R), in EBV-infected lymphoid cells. Here we demonstrate that R alone is sufficient to activate the pol promoter in EBV-negative B cells. Unlike other early promoters to which the R protein binds directly, its effect on the pol promoter does not appear to involve a direct DNA-binding mechanism. Instead, we found that two cellular transcription factors, an upstream stimulatory factor USF, and a member of the E2F family of proteins, bind directly to the pol promoter at positions ؊795 to ؊786 and ؊186 to ؊170, respectively, regions previously identified as important for activation of the pol promoter. These two sites contribute to or are essential for transactivation of the pol promoter by R in EBV-noninfected B cells. These data suggest that the R immediate-early protein may activate a key early EBV promoter (pol) through both USF and E2F. * Corresponding author. Phone: (919) 966-3036. Fax: (919) 966-3015.on July 10, 2020 by guest http://jvi.asm.org/ Downloaded from BMRF1, and BHRF1/BHLF1, R appears to act indirectly either together with or through E2F and USF. MATERIALS AND METHODSCell lines. Raji and Akata are latently EBV-infected B-lymphocyte lines. DG75 is an EBV-negative Burkitt's lymphoma B-cell line. NPC-KT is an EBVpositive epithelial cell line, and ADAH is an EBV-negative epithelial cell line. All lymphoid cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum. Epithelial cells were grown in Dulbecco's modified Eagle's medium plus 10% fetal calf serum.Plasmid constructs. The construction of plasmid pPOLCAT has been described previously (28). The internal deletion mutants were constructed by restriction digestion and religation. Site-directed mutants of pPOLCAT were made with the Bio-Rad Muta-Gene phagemid in vitro mutagenesis kit on the basis of the Kunkle method (53). The expression constructs of EBV immediate-early proteins have been described elsewhere (68). The pCMV-Z plasmid contains the BZLF1 cDNA fragment under the control of the cytomegalovirus immediateearly promoter in the pGem2-based vector (pHD1013). The pCMV-R plasmid is derived from a HindIII-HindIII fragment from a genomic expression construct, pCMV-RZ, such that the BRLF1 gene is linked to the cytomegalovirus immediate-early promoter in a pUC-18 vector.Transfections and chloramphenicol acetyltransferase assays. Plasmid DNA was purified through Qiagen columns. For each transfection, 10 7 cells were electroporated at 1,500 V with the University of Wisconsin Zapper electroporation unit. Cells were then suspended in 10 ml of RPMI 1640 medium supplemented with 10% fetal calf serum and incubated for 48 h at 37ЊC in 5% CO 2 . Cell extracts were prepared by washing cell pellets t...

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