Epstein-Barr Virus Transcription Activator R Upregulates BARF1 Expression by Direct Binding to Its Promoter, Independent of Methylation

Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) is considered a major viral oncogene in epithelial cells and has immune-modulating properties. However, in B cells and lymphomas, BARF1 expression is restricted to the viral lytic replication cycle. In this report, the transcriptional regulation of BARF1 during lytic replication is unraveled. Bisulfite sequencing of various cell lines indicated a high level of methylation of the BARF1 gene control region. A BARF1 promoter luciferase reporter construct was created using a CpG-free vector, enabling true assessment of promoter methylation. Induction of the EBV lytic cycle is mediated by the immediate-early proteins BZLF1 (Z) and BRLF1 (R). R was found to activate expression of the BARF1 promoter up to 250-fold independently of Z and unaffected by BARF1 promoter methylation. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and specific mutagenesis of the R-responsive elements (RREs) demonstrated direct binding of R to RREs between nucleotides -554 and -327 relative to the BARF1 transcriptional ATG start site. The kinetics of BARF1 expression upon transactivation by R showed that BARF1 mRNA was expressed within 6 h in the context of the viral genome. In conclusion, expression of the BARF1 protein during lytic replication is regulated by direct binding of R to multiple RREs in the gene control region and is independent of the promoter methylation status. The early kinetics of BARF1 upon transactivation by R confirm its status as an early gene and emphasize the necessity of early immune modulation during lytic reactivation.