The regions of the herpes simplex virus type 1 immediate early protein Vmwl75 required for site specific DNA binding closely correspond to those involved in transcriptional regulation

Abstract

The immediate-early (IE) protein Vmw175 (ICP4) of HSV-1 is required for the transcription of later classes of viral genes and the repression of IE gene expression. We have previously constructed a panel of plasmid-borne insertion and deletion mutants of the gene encoding Vmw175 and assayed their ability to regulate transcription in transient transfection assays. By this approach we have mapped the regions of the Vmw175 amino acid sequence that are required for transcriptional activation and repression of herpes virus promoters. This paper describes the use of nuclear extracts, made from cells transfected with these mutant plasmids, in gel retardation DNA binding assays in order to define the regions of Vmw175 involved in binding to a specific Vmw175 DNA binding site. The results show that amino acid residues 275-495 (a region which is highly conserved between Vmw175 and the varicella-zoster virus "IE" 140K protein) include structures which are critically required for specific DNA binding, transactivation and repression. This raises the interesting paradox that although the specific DNA sequence recognized by Vmw175 is not commonly found in its target promoters, the protein domain required for recognition of this sequence is required for promoter activation.