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We have examined the factors which influence the expression of a major 2.7-kilobase (kb) early transcript encoded by the long repeat of the human cytomegalovirus (HCMV) strain AD169 genome. Previously, by deletion analysis, we determined that the promoter for this early RNA consisted of multiple cis-acting elements (Klucher et al., J. Virol. 63:5334-5343, 1989). Using extracts prepared from HeLa cells as well as from infected and uninfected foreskin fibroblasts, we also obtained evidence for the interaction of a cellular factor with one of these elements. In this study, we have further defined the specificity and functional importance of this binding. On the basis of DNase I footprinting and methylation interference assays, we localized the site of interaction to a region (nucleotides -113 to -106 relative to the mRNA start site) which contains homology to the binding site for the adenovirus major late transcription factor (MLTF), also referred to as the upstream stimulatory factor (USF). The contact points of binding between the cellular factor and the guanine residues within this segment were consistent with the pattern of binding for USF/MLTF. Additionally, by using oligonucleotides containing the binding sites for USF/MLTF from the adenovirus major late promoter and the HCMV 2.7-kb RNA promoter as competitors in gel retardation assays, we were able to show that USF/MLTF bound to the two promoters with similar affinity. Correlation of the binding activity with in vivo functional importance was provided by mutagenesis and transient-expression assays. A point mutation within the HCMV USF/MLTF site lowered the affinity of binding 5- to 10-fold and decreased the inducible activity of the HCMV 2.7-kb RNA promoter by approximately 50%. Furthermore, the addition of the HCMV USF/MLTF site to a minimal 2.7-kb RNA promoter containing only the TATA sequence resulted in an increase in HCMV inducible transcriptional activity of 6- to 20-fold. However, the HCMV USF/MLTF site could not functionally substitute for the TATA sequence. These studies further support the idea that for maximal response to the HCMV infection, the 2.7-kb RNA promoter requires multiple cis-acting sequences, two of which include the binding sites for USF/MLTF and TFIID.

The human cytomegalovirus 2.7-kilobase RNA promoter contains a functional binding site for the adenovirus major late transcription factor