Identification and characterization of the protein product of gene 67 in equine herpesvirus type 1 strain Ab4.

Equine herpesvirus type 1 (EHV-1) strain Ab4 gene 67 has no counterpart in any herpesvirus sequenced to date. To identify and characterize the product of EHV-1 gene 67, we have expressed the putative amino acids 11 to 260 encoded by gene 67 as a beta-galactosidase fusion protein in Escherichia coli. The expressed fusion protein has been used to generate an antiserum raised against the gene 67 product. Immunoblotting and immunoprecipitation experiments have revealed that the anti-67 serum specifically recognizes a polypeptide with an M(r) of 36,000 (the 36K polypeptide) in infected cell extracts. The gene 67 protein is regulated as an early polypeptide in EHV-1 strain Ab4 infected cells and post-translational modification experiments have revealed that the protein is phosphorylated, but not glycosylated. The gene 67 protein has been transiently expressed in BHK-21/C13 cells using plasmid pCMV67, which contains the putative gene 67 ORF under the control of the cytomegalovirus immediate early promoter. Immunoblotting experiments with anti-67 have shown that the 36K protein is expressed at high levels in transfected cells. From both immunofluorescence and cellular fractionation experiments it is concluded that the gene 67 protein is associated with intracellular membranes and produces novel ribbon or filament-like structures within the cytoplasm of infected cells. We have demonstrated that the gene 67 product is a component of the virion nucleocapsid/tegument.