Replication and Transcription Activator (RTA) of Murine Gammaherpesvirus 68 Binds to an RTA-Responsive Element and Activates the Expression of ORF18

ABSTRACT The replication and transcription activator (RTA), mainly encoded by open reading frame 50, is an immediate-early gene product that is conserved among all characterized gammaherpesviruses. Previous studies have demonstrated that RTA proteins of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) can activate the promoter of many viral early lytic genes through direct or indirect mechanisms. Murine gammaherpesvirus 68 (MHV-68) is genetically related to KSHV and EBV, and the RTA homologue from MHV-68 also initiates the lytic cycle of gene expression. Although two RTA-dependent promoters had been identified in MHV-68, the mechanism of the interaction between RTA and the promoters was not characterized. In this study, we first identified an RTA-responsive promoter in the left origin of lytic replication region of MHV-68 through a reporter assay and mapped a 27-bp RTA-responsive element (RRE) through systematic deletions. Interestingly, sequence analysis identified a second RRE in this region. An electrophoretic mobility shift assay (EMSA) and a chromatin immunoprecipitation (ChIP) assay showed that RTA can bind directly to these two RREs in vitro or in vivo . Mutagenesis studies have further characterized the nucleotides important for mediating RTA binding by an EMSA. Moreover, we engineered RRE-deleted viruses and demonstrated in the context of the viral genome that one of the RREs mediates the RTA-dependent activation of an essential lytic gene, ORF18, during de novo infection. To our knowledge, this is the first time that RTA binding sites in MHV-68 have been identified. Since ORF18 regulates viral late gene expression, our study has also contributed to the delineation of the expression cascade of gammaherpesvirus lytic genes.