Date of Award
Doctor of Philosophy (PhD)
Ann E. Campbell
Timothy J. Bos
Julie A. Kerry
The HCMV UL98 early alkaline exonuclease gene promoter was examined to determine the DNA sequences as well as viral and/or cellular proteins functional in the regulation of this early gene. To assess promoter activation, UL98 promoter sequences were first cloned upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with expression plasmids which express the HCMV major immediate early (IE) proteins 1E72 and 1E86. To more specifically determine the importance of individual cis-acting elements in UL98 promoter activation, the promoter region underwent mutagenesis to delete or alter sequences. The variant promoters were again cloned into a reporter-CAT construct and analyzed in transient transfection assays to assess changes in promoter activity in response to viral or virally induced proteins.
Analysis of promoter activation indicated that the UL98 promoter required the presence of the IE72 and 1E86 immediate early (IE) proteins. In comparison to a prototypical early promoter which regulates the polymerase (pol) gene, UL98 promoter activation levels were equal to or even greater than that of pol in response to the IE transactivators. In the presence of all viral proteins, activation of the UL98 promoter continually increased when analyzed at 24, 48, and 72 hours.
Deletion analysis showed that a 13 by sequence located between −64 and −51 is required for UL98 promoter activation by IE proteins. Site-directed mutations generated in two cellular transcription factor binding sites resulted in a drastic reduction in promoter activation. A mutation in the cyclic AND response element (CRE) (−82 to −75) resulted in a 70% loss of promoter activation. The UL98 promoter was also poorly activated in the presence of a mutation generated in the gamma interferon response element (γIRE) (−37 to −30), a transcription factor binding site downstream of −51. Binding and competition experiments via gel mobility shift assays provided conclusive evidence that the CREB protein binds the CRE site in the UL98 promoter.
These data indicate that the UL98 early promoter is regulated primarily by the CRE and gamma IRE sequences. UL98 promoter activation therefore relies upon the presence of a combination of elements in their defined flanking positions.
Jervey, Tabmitha P..
"Regulation of the HCMV UL98 Promoter by Cooperation of the Cyclic AMP Response Element and Gamma Interferon Response Element"
(2001). Doctor of Philosophy (PhD), dissertation, , Old Dominion University, DOI: 10.25777/y0w4-6s06