Identification and characterization of the equine herpesvirus type 1 inverted repeat gene 4 (IR4) product

EHV-1 gene expression is coordinately regulated and sequentially ordered in an immediate-early, early, and late fashion. Previous work in our laboratory identified three regulatory genes termed immediate-early (IE), UL3, and ICPO. The protein products of these three genes trans-activate or trans-repress EHV-1 promoters in transient transfection assays.;The IR4 gene encodes a protein with homology to the herpes simplex virus type 1 ICP22 protein, an immediate-early protein that is involved in HSV-1 gene regulation. Use of IR4 antipeptide antiserum and TrpE-IR4 antiserum in immunoblot analyses revealed that the IR4 protein is first detected between 2 to 3 h postinfection, and is expressed as multiple proteins that migrate between 42 and 47 kDa. Indirect immunofluorescence, and laser scanning confocal microscopy studies revealed that IR4 proteins localize primarily to the nucleus and present a diffuse staining pattern. Immunoblot analyses of purified EHV-1 virions demonstrated the presence of IR4 proteins within the virion.;To determine whether the IR4 protein is involved in EHV-1 gene regulation, an IR4 expression construct was engineered and used in transient transfection assays. The results of these studies revealed that the IR4 protein: (i) in the absence of other EHV-1 proteins, does not efficiently trans-activate or trans-repress EHV-1 promoters, (ii) enhances upregulation of the IE promoter by the UL3 protein, (iii) enhances transactivation of early promoters by the IE protein, (iv) enhances the transactivation of early and late promoters by the IE and UL3 proteins, (vi) acts synergistically with the ICPO protein to trans-activate late promoters, and (vi) interacts synergistically with the IE gene product to trans-activate the heterologous HSV-1 ICP4 promoter.;Studies using glutathione S-transferase (GST)-IR4 fusion proteins indicated that the IR4 protein does not bind DNA in gel-shift assays; however, when the IR4 protein is added with the GST-IE protein, an EHV-1 DNA binding protein, a significant increase in DNA binding was observed compared to GST-IE alone. These results suggest that the IR4-IE synergistic effect on promoters seen in transient transfection assays may be the result of IR4 and IE interacting to increase the ability of IE to bind DNA.