Autostimulation of the Epstein-Barr virus BRLF1 promoter is mediated through consensus Spl and Sp3 binding sites

As an essential step in the lytic cascade, the Rta homologues of gammaherpesviruses all activate their own expression. Consistent with this biologic function, the Epstein-Barr virus (EBV) Rta protein powerfully stimulates the promoter of its own gene, Rp, in EBV-positive B cells in transient-transfection reporter-based assays. We analyzed the activity of RpCAT in response to Rta by deletional and site-directed mutagenesis, Two cognate Sp1 binding sites located at -279 and -45 relative to the transcriptional start site proved crucial for Rta-mediated activation. Previously described binding sites fur the cellular transcription factor Zif268 and the viral transactivator ZEBRA were found to be dispensable for activation of RpCAT by Rta, Gel shift analysis, using extracts of B cells in latency or induced into the lytic cycle, identified Sp1 and Sp3 as the predominant cellular proteins bound to Rp near -45, During the lytic cycle, ZEBRA bound Rp near the Sp1/Sp3 site. The binding of Sp1 and Sp3 to Rp correlated with the reporter activities in the mutagenesis study, establishing a direct link between transcriptional activation of Rp by Rta and DNA binding by Sp1 and/or Sp3. The relative abundance or functional state of the cellular Sp1 and Sp3 transcription Factors may be altered in response to stimuli that induce the BRLF1 promoter and thereby contribute to the activation of the viral lytic cycle.