Site-specific Binding of the Human Cytomegalovirus IE2 86-kilodalton Protein to an Early Gene Promoter

We have previously demonstrated that the human cytomegalovirus (HCMV) immediate-early region 2 86-kDa protein (the IE2 86 protein) is the major transactivator of the HCMV early promoter for the 2.2-kb class of RNAs (open reading frame UL 112-113). Here we show that specific stimulation of this promoter by IE2 86 in transient-expression assays requires sequences located between nucleotides (nt) -113 and -58 relative to the transcription start site; this is also the major regulatory region for this promoter during HCMV infection. To determine whether IE2 86 can bind to this promoter, a glutathione-S-transferase (GST)-IE2 86 fusion protein was incubated with the 32P-labeled promoter and specific binding was assessed by retention of the protein-DNA complex on glutathione-agarose beads. DNase I footprint analysis was also used to map the sequences involved in the binding. Our results indicate that three regions, located between nt -286 and -257, nt -248 and -218, and nt -148 and -120, bind strongly to the IE2 86 protein and share sequence similarity with the previously identified cis repression signal located near the cap site of the major HCMV IE gene. In addition, there is a weaker binding region between nt -113 and -85, which shares some sequence homology with the cis repression signal element and the strong binding regions of the 2.2-kb RNA promoter but lacks one of the two CG dinucleotides present in all of the high-affinity binding sites. With a set of IE2 86 protein deletion mutants, we also show that the DNA-binding domain spans a large region in the carboxy-terminal half of the protein.