CLONING AND EXPRESSION OF AN EQUINE HERPESVIRUS-1 ORIGIN-BINDING PROTEIN

Equine herpesvirus 1 (EHV-1) is an important pathogen of horses and is closely related to several important human pathogens, herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster vir us, The EHV-1 genome contains open reading frames similar in sequence to the HSV-1 replication genes. PCR was used to clone EHV-1 gene 53, which is similar in sequence to the HSV-1 UL9 gene. The gene 53 product has re gions of striking similarity to the HSV-1 UL9 and VZV gene 51 products . In vitro transcription and translation of this gene generated a prot ein of 87 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further characterization of this protein was accompl ished through the use of gel shift analysis. The in vitro-synthesized protein bound sequence specifically to EHV-1 Ori(s) as well as HSV-1 O ri(s). A series of triple-base-pair substitutions across the previousl y defined HSV-1 origin-binding protein consensus site was used in gel shift analysis to show that the EHV-1 origin-binding protein bound to the same consensus site as the HSV-1 origin-binding protein, 5'-CGTTCG CACTT-3'. Using a nuclear extract of EHV-1-infected RK13 cells, we hav e identified an activity that interacts similarly with this consensus site. In gel shift assays, the retarded band arising from the nuclear extract migrated similarly to the retarded band arising from in vitro- translated EHV-1 gene 53 An N-terminal deletion of EHV-1 gene 53 was a lso created, expressed in vitro, and used in gel shift assays to local ize the DNA-binding domain. Results of these experiments indicated tha t amino acids 1 to 499 were dispensable for binding and that the C-ter minal fragment (amino acids 500 to 888) recognized the same consensus site as did the wild-type protein. Thus, the product of EHV-1 gene 53 is an origin-binding protein with a high degree of similarity to the H SV-1 and varicella-zoster virus origin-binding proteins and possibly s erves as the initiator of DNA replication in EHV-1.