BINDING OF CELLULAR REPRESSOR PROTEIN OR THE IE2 PROTEIN TO A CIS-ACTING NEGATIVE REGULATORY ELEMENT UPSTREAM OF A HUMAN CYTOMEGALOVIRUS EARLY PROMOTER

We have previously shown that the human cytomegalovirus early UL4 prom oter has upstream negative and positive cis-acting regulatory,elements . In the absence of the upstream negative regulatory region, the posit ive element confers strong transcriptional activity. The positive elem ent contains a CCAAT box dyad symmetry and hinds the cellular transcri ption factor NF-Y. The effect of the negative regulatory element is ne gated by the viral IE2 protein (L. Huang, C. L. Malone, and M. F. Stin ski, J. Virol 68:2108, 1994). We investigated the binding of cellular or viral IE2 protein to the negative regulatory region. The major cis- acting negative regulatory element was located between -168 and -134 b p relative to the transcription start site. This element could be tran sferred to a heterologous promoter, and it functioned in either orient ation. Mutational analysis demonstrated that a core DNA sequence in th e cis-acting negative regulatory element, 5'-GTTTGGAATCGTT-3'; was req uired for the binding of either a cellular repressor protein(s) or the viral IE2 protein. The cellular DNA binding activity was present in b oth nonpermissive HeLa and permissive human fibroblast cells but more abundant in HeLa cells. Binding of the cellular repressor protein to t he upstream cis-acting negative regulatory element correlates with rep ression of transcription from the early UW promoter. Binding of the vi ral IE2 protein correlates with negation of the repressive effect.