IDENTIFICATION OF BINDING-SITES FOR THE 86-KILODALTON IE2 PROTEIN OF HUMAN CYTOMEGALOVIRUS WITHIN AN IE2-RESPONSIVE VIRAL EARLY PROMOTER

The 86-kDa IE2 protein (IE86) of human cytomegalovirus (HCMV) can act as both an activator and a repressor of gene expression. The mechanism s for both of these functions are not well defined. It has recently be en demonstrated that this protein has sequence specific DNA binding pr operties: it interacts directly with a target sequence that is located between the TATA box and the cap site of its own promoter. This seque nce, termed the CRS (cis repression signal) element, is required for n egative autoregulation of the IE1/IE2 enhancer/promoter by IE2. We dem onstrate now that binding of this protein to DNA is not confined to th is site but occurs also within an early promoter of HCMV that has prev iously been shown to be strongly IE2 responsive. By DNase I protection analysis using a purified, procaryotically expressed IE2 protein, we could identify three binding sites within the region of -290 to -120 o f the UL112 promoter of HCMV. Competition in DNase I protection experi ments as well as gel retardation experiments showed that the identifie d binding sites are specific and have high affinity. Deletion of IE2 b inding sites from this promoter reduced the level of transactivation; however, the remaining promoter could still be stimulated about 40-fol d. Constructs in which IE2 binding sites were fused directly to the TA TA box of the UL112 promoter did not reveal a significant contribution of these sequences to transactivation. However, if an IE2 binding sit e was reinserted upstream of nucleotide -117 of the UL112 promoter, an increase in transactivation by IE2 was obvious, whereas a mutated seq uence could not mediate this effect. This finding suggests that DNA-bo und IE2 can contribute to transactivation but seems to require the pre sence of additional transcription factors. Moreover, a comparison of t he detected IE2 binding sites could not detect a strong homology, sugg esting that this protein may be able to interact with a broad spectrum of different target sequences.