Identification and Characterization of the Protein Product of Gene 71 in Equine Herpesvirus 1

Abstract

Equine herpesvirus 1 (EHV-1) strain Ab4 gene 71 is predicted to encode a primary product with a M(r) of 80.1K. We have previously constructed a deletion/lacZ insertion mutant, ED71, and demonstrated that gene 71 is dispensable for growth of virus in cell culture. We have now constructed a gene 71 revertant, Re71. To identify and characterize the product of gene 71, we produced a specific antiserum, anti-71, against a beta-galactosidase fusion protein containing the carboxy terminus of the gene 71 polypeptide. Using the anti-71 serum, mutant ED71 and the revertant Re71, we have demonstrated that gene 71 encodes a 192K polypeptide. Experiments with glycosylation inhibitors revealed that the protein product of gene 71 is N-glycosylated and heavily O-glycosylated. When the 192K polypeptide is synthesized in the presence of monensin, the M(r) of the polypeptide is reduced to 80K, the predicted unmodified M(r) of the gene 71 polypeptide. The gene 71 product is found in virions and L particles in a fully processed form that runs as a diffuse band in electrophoresis, with a M(r) in excess of 200K. Immunofluorescence and virion surface labelling experiments showed that the polypeptide product of gene 71 is located on cellular membranes and the virion envelope. A time course of infection confirmed that gene 71 is regulated as a leaky late gene in infected cells. Finally, using wild-type EHV-1 Ab4, mutant ED71, revertant Re71 and two antibodies (P19 against EHV-1 glycoprotein gp300, and anti-71) we conclusively demonstrated that gene 71 encodes gp300. This contradicts published results with P19 alone, which indicated gp300 was the product of EHV-1 gene 28.