Date of Award
Doctor of Philosophy (PhD)
Julie A. Kerry
Timothy J. Bos
Ann E. Campbell
Kenneth D. Somers
Gene expression during productive infection by the human cytomegalovirus (HCMV) occurs in an ordered and sequential manner, beginning with immediate early (IE), then early (E) and finally late (L) gene expression. Significant work has addressed the regulation of IE and E gene expression while relatively little work has addressed the control of late gene expression. In order to further address HCMV late gene expression, the promoter of the HCMV UL75 (glycoprotein H, gH) late gene was characterized. The data obtained in this study were combined with observations made in two other studies that have addressed HCMV late gene expression to develop a model of the regulation of HCMV late gene expression.
The gH promoter and numerous promoter mutants were cloned into a reporter vector to address sequences responsible for the regulation of gene expression. These gH promoter constructs were transfected into human fibroblasts and subsequently infected with HCMV. Our data revealed that viral infection was necessary to activate expression from the gH promoter, confirming observations made in other HCMV late gene studies. Deletion analysis of promoter sequences revealed that sequences upstream of the TATA element in the gH promoter are not necessary for wild type levels of gene expression. Further deletion of sequences downstream of the mRNA cap site (+1) revealed the presence of upstream control elements that were masked by the dominant downstream activation domain. Mutational analysis demonstrated that a PEA3 element present in this downstream domain is important for promoter activation. In addition, gel shift analysis revealed direct protein binding to the PEA3 element. Deletion and mutational analysis also revealed that multiple sequence elements between −38 and +14 are responsible for gH promoter activation. Gel shift analysis using promoter sequences from this region as probes and competitors revealed protein binding to a DAS element at +1 to +6. Despite direct protein binding, transfection analysis in fibroblasts revealed that the DAS element was not important for promoter activation, however it may be important in other cell types. Gel shift analysis also demonstrated a protein binding to promoter sequences between +1 and +35 at late times of infection that is likely involved in activation. This analysis reveals the complexity of gH promoter regulation as well as demonstrating similarities between all three HCMV late gene promoters characterized thus far.
McWatters, Bernard J..
"Characterization of the Human Cytomegalovirus UL75 Late Gene Promoter"
(2001). Doctor of Philosophy (PhD), Dissertation, Biological Sciences, Old Dominion University, DOI: 10.25777/n6az-e304