Transcriptional regulation of a human cytomegalovirus early gene in the context of the viral genome

The human cytomegalovirus (HCMV) early UL4 promoter has served as a useful model for studying the activation of early viral gene expression. Previous transient-transfection experiments detected cis-acting elements (NF-Y site and site 2) upstream of the transcriptional start site (L. Huang, and M. F. Stinski, J. Virol. 69: 7612–7621, 1995). The roles of two of these sites, the NF-Y site and site 2, in the context of the viral genome were investigated further by comparing mRNA levels from the early UL4 promoter in human foreskin fibroblasts infected by recombinant viruses with either wild-type or mutant cis-acting elements. Steady-state mRNA levels from the UL4 promoter with a mutation in the NF-Y site were comparable to that of wild-type. A mutation in an Elk-1 site plus putative IE86 protein binding sites decreased the steady-state mRNA levels compared to the wild-type at early times after infection. Electrophoretic mobility shift assays and antibody-supershifts detected the binding of cellular transcription factor Elk-1 to site 2 DNA with infected nuclear extracts, but not with mock infected nuclear extracts. The roles of the Elk-1 binding site alone in the context of the viral genome were investigated further by comparing expression levels from the early UL4 promoter in human foreskin fibroblasts infected with recombinant viruses containing either wild-type or mutant cis-acting elements. A site-specific mutation in the Elk-1 binding site resulted in an approximately 50% decrease in expression levels compared to the wild-type at early times after infection in HFFs. A series of recombinant viruses that contain various deletions of the UL4 promoter were also constructed and the roles of additional regulatory elements for the UL4 promoter activation were determined. The sequences between −102 and −50 were also critical for activation of the UL4 promoter at early times after infection. Viral gene expression was regulated by upstream cis-acting sites and by basic promoter elements that respond to the mitogen-activated protein kinase (MAPK) signal transduction pathways. Inhibitors of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway and the p38 MAPK pathway affected expression from the early viral UL4 promoter in the viral genome. Maximum activity of this early viral promoter requires sites for cellular transcription factors that are known to be activated by the MAPK/ERK or the p38 MAPK pathways plus viral or cellular factors that interact with the TATA-box and associated factors.