NEGATIVE REGULATION OF A HETEROLOGOUS PROMOTER BY HUMAN CYTOMEGALOVIRUS IMMEDIATE-EARLY PROTEIN IE2

The HCMV IE2 protein promiscuously activates transcription of many vir al and cellular genes. IE2 also negatively autoregulates its own expre ssion by binding to a strategically positioned IE2 binding site, calle d CRS, located immediately downstream of the TATA box of the HCMV majo r IE promoter. Here we show that IE2 is able to repress transcription driven by a heterologous promoter, RSV LTR. Repression of RSV LTR by I E2 is completely dependent of DNA sequences downstream of the TATA box of RSV LTR. A DNA sequence, 5'-CGATACAATAAACG-3', evidently matching the proposed CRS consensus sequence, is located between nucleotides -1 3 and +1 (relative to the transcription start site) of RSV LTR. Three lines of evidence support the notion that this RSV CRS element is invo lved in the IE2-mediated repression of RSV LTR. First, introduction of mutation to the RSV CRS element renders to the mutant RSV LTR resista nce to IE2-mediated repression. Second, a mutant IE2 defective in DNA binding cannot downregulate transcription from RSV LTR. Third, IE2 spe cifically binds to the wild-type, but not the mutant, RSV CRS element in vitro. These data, in conjunction with previous works, demonstrate that IE2 can passively repress transcription of homologous and heterol ogous promoters that contain a CRS element. (C) 1997 Academic Press.