Characterization of Binding Properties of the Immediate-Early Gene Product of Equine Herpesvirus Type 1

Abstract

The equine herpesvirus type 1 (EHV-1) immediate-early (IE) gene encodes a phosphoprotein that is essential for the activation of transcription from viral early and late promoters and that regulates the transcription from its own promoter. Employment of EHV-1 IE promoter DNA probes and glutathione S-transferase fusion proteins harboring truncated portions of the IE gene product in gel shift assays, super shift assays with anti-IE monoclonal antibodies, and DNase I footprinting analyses revealed: (1) amino acid residues 422 to 597 within the 1487-amino-acid IE protein are sufficient for sequence-specific DNA binding; (2) the IE protein binds to EHV-1 DNA at sequences from -11 to +14 that overlap the transcription initiation site (+1); (3) the conserved pentanucleotide 5′-ATCG- 3′ in the IE promoter located at nucleotides (nt) -6 to -2, relative to the transcription initiation site (+1), is critical for IE protein binding; (4) a weak binding site for the IE protein is also present at nt -92 to -82 of the IE gene within the sequence (-86)ATCGA(-82) in which four of the five nt in the consensus binding sequence are conserved; (5) the IE protein binds to sequences in EHV-1 early and late promoters that contain a degenerate version of the consensus sequence 5′-ATCGT-3′ and (6) mutation of the C or G nt in the pentanucleotide 5′-ATCGT-3′ prevents sequence-specific binding of the IE protein, whereas mutation of each of the other three nt only reduces binding. These results suggest that the IE protein can recognize the sites which differ slightly from the proposed consensus sequence. Overall, these findings suggest that formation of a specific complex between an IE protein and its own gene promoter may be a common mechanism used by Alphaherpesvirinae to autoregulate transcription of an essential IE gene. In addition, the finding that the DNA binding domain of the IE protein maps within amino acids 422 to 597, a domain conserved in the IR2 early protein that is a truncated form of the IE protein, suggests that the IR2 protein plays a role in the regulation of the IE gene expression.