The IE2 gene products of human cytomegalovirus specifically down-regulate expression from the major immediate-early promoter through a target sequence located near the cap site

The 82-kDa IE2 protein of human cytomegalovirus (HCMV) acts as both a powerful nonspecific trans activator of heterologous promoters and a negative autoregulator of HCMV immediate-early gene expression In assays. We show here that the highly specific down-regulationeffect occurs in permissive diploid as well as in nonpermissive vero cell and that the target sequences are conserved within the major immediate-early promoters of both HCMV and siman cytomegalovirus. The response sequences were localized between -67 +30 in the simian cytomegalovirus IE94 promoter of position +9 in the HCMV IE68 promoter. Deletion of sequences downstream of -14 in a target IE68-CAT gene the negative phenotype andresulted in a reporter gene that was stimulated instead of by inhibited by cotransfection with IE2 effector DNA. Insertion of an oligonucleotide containing from between -17 +9 into the IE68-CAT deletion construction restored autoregulation in either orientation. Furthermore, the full down-regulation phenotype when inserted at +10 into the nonresponsive IE175 promoter from herpes virus. Therefore, a specific response signal that at the DNA level must lie within these boundaries. Additional analysis with inserted oligonucleotides containing deletions or point mutations revealed that essential components of the signal lie between position -12 and +5. Therefore, negative autoregulation by HCMV IE2 in DNA cotransfection systems resembles that for simian virus 40 large T antigen and herpes simplex virus IE175 by acting through a signal located near the cap site, but the target sequence itself bears no resemblance to those utilized in these other viral systems.