Regulation of expression from the M142 promoter of mouse cytomegalovirus

Human cytomegalovirus (HCMV) is a common cause of infection with serious illness in immune-compromised people. HCMV can only infect humans, limiting the studies done with this virus. Animal viruses, such as mouse cytomegalovirus (MCMV) are useful models for HCMV. Despite extensive use as a model, very little is known about gene regulation in MCMV compared to HCMV. Hence, the goal of this research was to expand the knowledge of promoter regulation in MCMV. We examined regulation of an essential MCMV gene, m142. The m142 gene s closest homologs are HCMV IRS1 and TRS1 which have similar expression and function. Previous analysis showed that m142 is an immediate early gene, meaning cellular factors are sufficient for activation, although viral infection increased expression. However, neither the sequences required nor the regulatory factors were identified. Using a series of sequential deletion mutants of the m142 promoter controlling a secreted alkaline phosphatase (SEAP) reporter, we have identified important regulatory regions and some probable regulators. We found that a sequence between -901 and -875 nucleotides upstream of the transcription start sites was required for activation in the absence of viral infection. Further analysis by EMSA indicated involvement of a consensus Elk-1 site and the binding of Elk-1 protein in this region. Two important viral transcriptional regulators are IE1 and IE3. Co-transfection with plasmids expressing these proteins showed that IE1 and IE3 separately activated via distinct regions of the promoter, but also co-operated. Comparison of the co-transfections with infection indicated that additional viral factors likely regulate the m142 promoter via sequences between -713 to -579. We were able to narrow down the potential viral genes involved by inhibition of a subset of viral proteins, known as late genes, which were not required for this regulation. Finally, deletion of sequences between -875 and -713 resulted in an increase in promoter activity. As this was detected not only in the context of viral infection, but also when either IE1 or IE3 were co-transfected, a cellular regulator is likely involved in this repression. In summary, we have found that like immediate early promoters of HCMV, m142 is a long promoter with complex regulatory mechanisms.