CELLULAR OR VIRAL PROTEIN-BINDING TO A CYTOMEGALOVIRUS PROMOTER TRANSCRIPTION INITIATION SITE - EFFECTS ON TRANSCRIPTION

We have previously shown that the IE2 protein of human cytomegalovirus (CMV) represses its own synthesis by binding to the major immediate-e arly promoter (M, P. Macias and M, F. Stinski, Proc. Natl. Acad. Sci. USA 90:707-711, 1993). The binding of a viral protein (IE2) and a cell ular protein in the region of the transcription start site was investi gated by site-specific mutational analysis and electrophoretic mobilit y shift assay, The viral protein and the cellular protein require diff erent but adjacent core DNA sequence elements for binding, In situ che mical footprinting analysis of DNA-protein interactions with purified CMV IE2 protein or HeLa cell nuclear extracts demonstrated binding sit es that overlap the transcription start site. The IE2 protein footprin t was between bp -15 and +2, relative to the transcription start site, and the cellular protein was between bp -16 and +7, The ability of th e unknown human cellular protein of approximately 150 kDa to bind the CMV major immediate-early promoter correlates with an increase in the level of transcription efficiency. Mutations in the core DNA sequence element for cellular protein binding significantly reduced the level o f in vitro transcription efficiency. Mutations upstream and downstream of the core sequence moderately reduced the transcription efficiency level. Negative autoregulation of the CMV promoter by the viral IE2 pr otein may involve both binding to the DNA template and interference wi th the function of a cellular protein that binds to the transcription start site and enhances transcription efficiency.