STRUCTURAL AND ANTIGENIC IDENTIFICATION OF THE ORF12 PROTEIN (ALPHA-TIF) OF EQUINE HERPESVIRUS-1

The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex Virus t ype 1 (HSV-1) tegument phosphoprotein, alpha TIF (Vmw65; VP16), was id entified previously as the product of open reading frame 12 (ORF12) an d shown to transactivate immediate early (IE) gene promoters. However, a specific virion protein corresponding to the ORF12 product has not been identified definitively. In the present study the ORF12 protein, designated ETIF, was identified as a 60-kDa virion component on the ba sis of protein fingerprint analyses in which the limited proteolysis p rofiles of the major 60-kDa in vitro transcription/translation product of an ORF12 expression vector (pT7-12) were compared to those of puri fied virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 Virions and subvirion fractio ns using polyclonal antiserum and monoclonal antibodies generated agai nst a glutathione-S-transferase-ETIF fusion protein. Northern and West ern blot analyses of EHV-l-infected cell lysates prepared under Variou s metabolic blocks indicated that ORF12 is expressed as a late gene, a nd cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-I pr otein species suggested that ETIF and HSV-I alpha TIF are antigenicall y related. Last, DNA band shift assays used to assess ETIF-specific co mplex formation indicated that ETIF participates in an infected cell p rotein complex with the EHV-I IE promoter TAATGARAT motif. (C) 1997 Ac ademic Press.