Synthesis and processing of equine herpesvirus type 1 glycoprotein 14.
Glycoprotein 14 (gp14) of equine herpesvirus type 1 (EHV-1), the homolog of herpes simplex virus (HSV) glycoprotein B (gB), was investigated employing a panel of monoclonal antibodies to ascertain the regulatory class, rate of synthesis, and type of glycosylation of this polypeptide. Application of immunoprecipitation, Western blot, and SDS-PAGE analysis in conjunction with the use of metabolic inhibitors (cycloheximide, antinomycin D, phosphonoacetic acid, tunicamycin, and monensin), and time-course and pulse-chase experiments revealed the following information: (1) Three gp14-related polypeptides with molecular weights of 138 kilodaltons (K), 77-75K, and 55-53K are present in EHV-1-infected cell extracts. (2) All three species are synthesized in the presence of the DNA synthesis inhibitor phosphonoacetic acid although their synthesis is enhanced by DNA replication, indicative of a beta-gamma class molecule. (3) The 138K species is synthesized first as a precursor of the smaller species of gp14, the 77-75K and 55-53K forms. (4) Use of glycosylation inhibitors and digestion of immunoprecipitated gp14 with endoglycosidases indicate that the primary translation product is a 118K molecule which is cotranslationally glycosylated to the 138K form by the addition of high mannose oligosaccharides. (5) The 77-75K species contains both high mannose and hybrid oligosaccharides while the 55-53K form of gp14 contains some complex oligosaccharides. (6) In the absence of a reducing agent, the 138K polypeptide and a large 145K species are observed in both infected cell extracts and purified virions. Thus, EHV-1 gp14 appears to be synthesized as a large precursor molecule of 138K and is proteolytically cleaved to two smaller forms, 77-75K and 55-53K, which are linked by a disulfide bond(s) to form a 145K complex. This model of gp14 synthesis and maturation is similar to those proposed for a number of HSV gB equivalents found in the Alphaherpesvirnae.
Sullivan
D C
DC
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130-3932.
Allen
G P
GP
O'Callaghan
D J
DJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D006031:Glycosylation; D006564:Herpesviridae / Q000378:metabolism*; D004861:Herpesvirus 1, Equid / Q000378:metabolism*; D007106:Immune Sera / Q000276:immunology; D007700:Kinetics; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D014764:Viral Proteins / Q000096:biosynthesis* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D007106:Immune Sera; D014764:Viral Proteins