The equine herpesvirus 1 (EHV-1) immediate-early (IE) phosphoprotein is essential for the activation of transcription from viral early and late promoters and regulates transcription from its own promoter. The EHV-1 EICP22 protein, a homolog of ICP22 of herpes simplex virus, increased the in vitro DNA binding activity of the IE protein for sequences in the IE, early, and late promoters. The EICP22 protein affected the rate as well as the extent of the IE protein binding to promoter DNA sequences. To study the DNA binding activity of the IE protein, Trp493, Gln495, Asn496, and Lys498 of the WLQN region, which is directly involved in DNA binding, were replaced with Ser (IEW493S), Glu (IEQ495E), Ile (IEN496I), and Glu (IEK498E), respectively. Gel shift assays revealed that the glutathione S-transferase (GST)-IEQ495E(407-615) and GST-IEK498E(407-615) proteins failed to bind to the IE promoter, indicating that the Gln and Lys residues are important for the DNA binding activity. In the presence of the GST-EICP22 protein, DNA binding activity of the GST-IEQ495E(407-615) protein was restored, suggesting that the EICP22 protein cooperates with the IE protein to regulate EHV-1 gene expression. Transient-transfection assays also showed that the EICP22 protein allowed the IEQ495E mutant to be functional as a transactivator. These results are unique and may represent an important role for the EICP22 protein in EHV-1 gene regulation.