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.