Bovine coronavirus spike glycoprotein: localization of an immunodominant region at the amino-terminal end of S2

Abstract

We have identified the binding site of monoclonal antibodies (MAbs) against the $2 subunit of the bovine coronavirus spike (S) glycoprotein. The location of this site was first investigated by using prokaryotic expression of DNA restriction fragments covering the entire S gene. The amino acid sequence containing the antibody binding site was shortened from 70 to 20 amino acids by digestion of plasmid DNA with exonuclease III, followed by sequencing of the smallest digestion product encoding an immunoreactive fusion protein. Finally we synthesized a set of nonapeptides covering the 20 amino acid sequence extending from the N-terminal residue of the $2 subunit (Ala 769 to Tyr 798). MAbs reacted mainly with six consecutive overlapping peptides with the sequence TTGYRFTN-FEPFTV. Polyclonal antibodies from hyperimmunized or convalescent animals reacted only with the recombinant proteins identified by MAbs, and the hyperimmune serum bound to the same set ofpeptides. This suggests that this highly conserved linear antigenic determinant corresponds to an immunodominant region. This region resembles both in location and immunodominance the linear determinant defined on the infectious bronchitis virus $2 subunit. The presence of similar regions in the N-terminal region of the $2 subunit of other coronaviruses is discussed.

Simultaneous Detection of Antibodies to Mouse Hepatitis Virus Recombinant Structural Proteins by a Microsphere-Based Multiplex Fluorescence Immunoassay

“…Because the S protein is too large to be produced efficiently in E. coli, we prepared truncated molecules of the S protein, including the N-terminal S1 subunit, the C-terminal S2 subunit, and the middle part, designated Smid. In previous studies of MHV (3,16), bovine coronavirus (22), and severe acute respiratory syndrome coronavirus (15,26), this middle region was identified as an immunodominant linear neutralization domain by analysis using a prokaryotic protein expression system and monoclonal antibodies or patients' sera. As a result, large amounts of the His-tagged S1, S2, and Smid proteins were produced as insoluble proteins and were solubilized in a high-molar urea solution.…”

Antigenic variation among recent Japanese isolates of bovine coronaviruses belonging to phylogenetically distinct genetic groups

“…To identify the viral protein recognized by mAb 4H4, the binding of 4H4 in western blotting assays was examined. The assays showed that in the absence of 2-ME, the mAb 4H4 bound to a 105-kDa protein, which was identified as the S1 subunit of the S protein [34]. In the presence of 2-ME, the mAb 4H4 did not bind to any antigen (Fig.…”

Evolutionary dynamics of bovine coronaviruses: natural selection pattern of the spike gene implies adaptive evolution of the strains

“…The putative receptor-binding domain (so called C-domain spanning amino acid residues 326-540) is coloured blue and green. of the S1 subunit of BCoV and the N-terminus of the S2 subunit spanning amino acid residues 769-798 have been previously recognized using mAbs (Vautherot et al, 1992a;Yoo et al, 1991b). A polymorphic region spanning amino acid residues 456-592 has also been shown by sequence analysis of BCoV strains (Rekik & Dea, 1994).…”