IDENTIFICATION OF POSITIVE AND NEGATIVE REGULATORY REGIONS INVOLVED IN REGULATING EXPRESSION OF THE HUMAN CYTOMEGALOVIRUS UL94 LATE PROMOTER - ROLE OF IE2-86 AND CELLULAR P53 IN MEDIATING NEGATIVE REGULATORY FUNCTION

The human cytomegalovirus (HCMV) UL94 gene product is a herpesvirus-co mmon virion protein that is expressed with true late kinetics. To iden tify the important cis- and trans-acting factors which contribute to U L94 transcriptional regulation, we have cloned, sequenced, and analyze d UL94 promoter function by transient transfection analysis. Transfect ion of UL94 promoter-reporter gene constructs into permissive human fi broblasts or U373(MG) cells indicated that promoter activity was detec ted following infection with HCMV. Point mutations within a TATA-like element located upstream of the RNA start site significantly reduced U L94 promoter activity. Deletion mutagenesis of the promoter indicated that a positive regulatory element (PRE) was likely to exist downstrea m of the UL94 mRNA start site, while a negative regulatory element (NR E) was present upstream of the TATA box. At late times of infection, t he PRE appeared to have a dominant effect over the NRE to stimulate ma ximum levels of UL94 promoter activity, while at earlier times of infe ction, no activity associated with the PRE could be detected. The NRE, however, appeared to cause constitutive down-regulation of UL94 promo ter activity. Binding sites for the cellular p53 protein located withi n the NRE appeared to contribute to NRE function, and NRE function cou ld be recapitulated in cotransfection assays by concomitant expression of p53 and HCMV IE2-86 protein. Our results suggest a novel mechanism by which the cellular protein p53, which is involved in both transcri ptional regulation and progression of cellular DNA synthesis, plays a central role in the regulation of a viral promoter which is not activa ted prior the onset of viral DNA replication.