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 type 1 (HSV-1) tegument phosphoprotein, alpha TIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12) and 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 basis of protein fingerprint analyses in which the limited proteolysis profiles of the major 60-kDa in vitro transcription/ translation product of an ORF12 expression vector (pT7-12) were compared to those of purified virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 virions and subvirion fractions using polyclonal antiserum and monoclonal antibodies generated against a glutathione-S-transferase-ETIF fusion protein. Northern and Western blot analyses of EHV-1-infected cell lysates prepared under various metabolic blocks indicated that ORF12 is expressed as a late gene, and cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-1 protein species suggested that ETIF and HSV-1 alpha TIF are antigenically related. Last, DNA band shift assays used to assess ETIF-specific complex formation indicated that ETIF participates in an infected cell protein complex with the EHV-1 IE promoter TAATGARAT motif.
Lewis
J B
JB
Department of Oral Biology/Microbiology, Medical College of Georgia, Augusta 30912-1126, USA.
Thompson
Y G
YG
Feng
X
X
Holden
V R
VR
O'Callaghan
D
D
Caughman
G B
GB
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000378:metabolism*; D004861:Herpesvirus 1, Equid / Q000235:genetics / Q000378:metabolism*; D006736:Horses; D016366:Open Reading Frames; D012333:RNA, Messenger / Q000032:analysis; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D000956:Antigens, Viral; D017405:Herpes Simplex Virus Protein Vmw65; D012333:RNA, Messenger; D014764:Viral Proteins