Binding of SP1 to the immediate-early protein-responsive element of the human cytomegalovirus DNA polymerase promoter

Abstract

Human cytomegalovirus (HCMV), a member of the herpesvirus family of DNA viruses, encodes two major immediate-early (IE) transcription factors, IE72 and IE86, that are important for regulated expression of the viral genome. The purpose of this study was to identify the host cellular components required for regulation of the HCMV DNA polymerase promoter (UL54) by HCMV IE proteins. Extensive mutagenesis defined a DNA element located between -54 and -43 relative to the transcription start site that was required for both basal transcriptional activity and transactivation by viral IE proteins. A single copy of the UL54 -54/-43 sequence enhanced the responsiveness of a heterologous minimal promoter to HCMV IE proteins. Fractionation of extracts prepared from uninfected cells led to the isolation of two cellular proteins with apparent molecular masses of 95 and 105 kDa that bound specifically to the UL54 -54/-43 element. Biochemical and immunochemical analyses identified this protein as the transcription factor SP1. Although initial inspection of the UL54 -54/-43 sequence did not predict an SP1 binding site, subsequent analyses indicated that it is indeed a nonconsensus GC box. We propose that SP1 is required to direct basal levels of promoter activity and that SP1-regulated transcription complexes allow the entry of HCMV IE proteins into the transcription cycle.