Structural analysis of the conformational domains involved in neutralization of bovine coronavirus using deletion mutants of the spike glycoprotein S1 subunit expressed by recombinant baculoviruses.
Two conformation-dependent neutralizing epitopes, A and B, have been mapped to the S1 subunit of the S spike glycoprotein of bovine coronavirus (BCV). In order to characterize the structure of these antigenic sites, we constructed a series of cDNA clones encoding deleted or truncated S1 derivatives and expressed the modified genes in insect cells using recombinant baculoviruses. Monoclonal antibodies directed against epitopes A and B recognized only the mutant S1 polypeptides containing amino acids 324-720, as demonstrated by immunoprecipitation and Western blot analysis in the absence of beta-mercaptoethanol. In addition, two domains within this region were identified and only mutants containing both domains were immunoreactive, indicating that both were critical in the formation of the antigenic determinants. One domain was localized between residues 324 and 403 and the other at residues 517-720. Deletion of either domain inhibited extracellular secretion of the mutant proteins whereas mutants containing both or none of the domains were secreted efficiently. This observation suggests a vital function of the native conformation of the S1 protein in both antigenic structure and intracellular transport. Antigenic determinants A and B were not distinguished, but these determinants appeared to require both domains for epitope formation. Our results suggest that the antigenic determinants formed by two domains are likely associated with the probable polymorphic region of the BCV S1 subunit.
Yoo
D W
DW
Veterinary Infectious Disease Organization, Saskatoon, Saskatchewan, Canada.
Parker
M D
MD
Song
J
J
Cox
G J
GJ
Deregt
D
D
Babiuk
L A
LA
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics*; D001665:Binding Sites; D002417:Cattle; D002872:Chromosome Deletion; D003332:Coronaviridae / Q000235:genetics* / Q000276:immunology; D006031:Glycosylation; D008562:Membrane Glycoproteins; D009036:Moths / Q000382:microbiology; D009154:Mutation; D009500:Neutralization Tests; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*
D000956:Antigens, Viral; D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C064484:spike protein S, Transmissible gastroenteritis virus