An in vitro system for human cytomegalovirus immediate early 2 protein (IE2)-mediated site-dependent repression of transcription and direct binding of IE2 to the major immediate early promoter

Abstract

In vivo, negative autoregulation of the strong major immediate early promoter (MIEP) of human cytomegalovirus requires the viral immediate early 2 protein (IE2) and a cis element located from position -13 through position -1 relative to the transcription start site. We have established an in vitro transcription system that reproduces the specificity of IE2-mediated negative autoregulation. The carboxyl-terminal 290-amino acid fragment of IE2 was purified as a bacterial fusion protein. Addition of this chimeric protein to the cell-free system specifically repressed transcription from the MIEP containing the wild-type cis-acting repressor element but not from a mutated template in which the cis element had been replaced by heterologous DNA. Control protein and a mutant IE2 fusion protein containing two specific amino acid substitutions in a putative zinc finger motif did not repress the MIEP in vitro. Using conditions defined by this functional assay, we demonstrated by mobility-shift experiments that IE2 binds directly and specifically to DNA bearing the cis-acting repressor element. In addition, IE2 bound to the MIEP in the in vitro transcription reaction mixture.