Characterization of glycoprotein B of the gammaherpesvirus equine herpesvirus-2.
Twenty-two monoclonal antibodies (MAbs) were generated to the gammaherpesvirus equine herpesvirus-2 (EHV-2). Using Western blot analysis, eight MAbs recognized an Escherichia coli glutathione S-transferase (GST)-glycoprotein B (gB) fusion protein and, using overlapping GST-gB fusion proteins, a neutralization epitope was mapped to amino acids 29-74. One of the gB-specific MAbs was used to characterize the glycosylation and kinetics of synthesis of EHV-2 gB. EHV-2 gB is synthesized as a 97 kDa polypeptide that is co-translationally modified to a 130 kDa high-mannose precursor that forms a 260 kDa dimer shortly after synthesis. Each 130 kDa precursor is endoproteolytically cleaved to disulphide-linked subunits of 75 and 58 kDa prior to further processing to complex oligosaccharide-containing subunits of 89 and 65/62 kDa. The 89 and 65/62 kDa subunits of EHV-2 gB contain 39 and 17 kDa of N-linked oligosaccharides, respectively, and do not contain any O-linked oligosaccharides. Western blot analysis of purified EHV-2 virions established that gB exists as a 320 kDa dimer in the virion envelope.
Centre for Equine Virology, The University of Melbourne, School of Veterinary Science, Parkville, Victoria, Australia. sholloway@angis.usyd.edu.au
S A
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Holloway
M J
MJ
Studdert
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Drummer
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D018397:Equidae; D018142:Gammaherpesvirinae / Q000276:immunology*; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000378:metabolism; D009500:Neutralization Tests; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014415:Tunicamycin / Q000494:pharmacology; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014771:Virion
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D018985:Epitopes, B-Lymphocyte; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D014415:Tunicamycin; D006026:Glycoside Hydrolases; D009439:Neuraminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase