B00009Neutralizing Epitopes for Bovine Coronavirus are Associated with the Hypervariable Domain in the S1 Spike Glycoprotein
Dongwan Yoo (1), Hana Weingartl (1), and Dirk Deregt (2)
(1) Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1 and (2) Animal Disease Research Institute, Lethbridge, AB, Canada T1J 3Z4
Abstract:
Two conformation-dependent neutralizing epitopes have been previously identified in the S1 subunit of the spike glycoprotein of bovine coronavirus. Using neutralizing monoclonal antibodies and a series of deletion mutants of the recombinant S1 proteins expressed in baculovirus, we found two domains important for the formation of antigenic determinants (Yoo et al., Virology 183: 91, 1991). In order to further characterize the antigenic structure of the S1 subunit protein, we generated mutant coronaviruses resistant to neutralization by group A monoclonal antibody. The S1 gene was cloned from the mutant coronaviruses, and the nucleotide sequence was determined. By comparisons of the mutant sequences with the wild-type sequence, we identified a single nucleotide change at position 1583 of the S1 gene. This single point mutation resulted in the change of alanine residue to valine residue, demonstrating that alanine at position 528 of the S1 protein is critical for the group A antigenic formation. The residue lies in the antigenic domain II as predicted previously and is associated with the probable hypervariable domain of bovine coronavirus, indicating that the hypervariable domain of the bovine coronavirus spike glycoprotein contains major antigenic sites.
Introduction:
The genome of bovine coronavirus (BCV) is a single-stranded RNA with positive polarity of approximately 30 Kb and encodes four major structural proteins; nucleocapsid protein, membrane-associated protein, hemagglutinin-esterase protein, and spike protein. The spike (S) protein is glycosylated and the major viral component associated with the virion membrane. The S protein possesses functions responsible for cell-binding, cell fusion, and induction of neutralizing antibody response. The BCV S glycoprotein is cleaved post-translation at amino acids positions 768-769, producing S1 and S2 two subunits. Accumulated information indicates that the carboxyl-terminal S2 subunit is an integral membrane protein comprising the stalk portion of the peplomer whereas the amino-terminal S1 subunit constitutes the bulbous part of the peplomer structure (for a review, see 1).
Two groups of non-overlapping antigenic sites responsible for BCV neutralization have been previously identified using conformation-dependent, neutralizing monoclonal antibodies (2). These epitopes have been mapped to the S1 portion of the S protein in BCV using the recombinant proteins expressed in baculovirus (3). Subsequent studies using a series of deleted and truncated mutant S1 derivatives expressed in baculovirus have identified that two domains in the S1 portion are closely associated with these antigenic sites (4). It appears that these domains overlap with the probable polymorphic region identified in mouse hepatitis virus strain 4 and its neuroattenuated and neutralization resistant variants (5). Thus, the current study was conducted to further characterize relationship of the BCV neutralizing epitopes and the probable polymorphic region present in the BCV S1 subunit.
Materials and Methods:
Quebec strain of BCV was propagated in MDBK cells in this study. Monoclonal antibody resistant mutant viruses were generated by incubating approximately 100 PFU of BCV with 1:100 dilution of the group A neutralizing monoclonal ascites HB10-4 (2) for 60 min at 37 C. The reaction was plated on MDBK cells with 1% agarose overlay, and plaques were selected. The plaque-picked virus was then amplified for two subsequent passages in the presence of HB10-4. For cDNA cloning of the viral RNA, total cellular RNA was extracted by standard methods from cells infected with virus. Two primers were designed to represent nucleotide positions 946-970 (upstream primer: 5'-CAGCCAATTGCAGATGTTTACCGAC-3') and 2187-2161 (downstream primer: 5'-ACTATCAAAATAGTTAATAGGTTGCAG-3'). The PCR products were cloned into SmaI site of plasmid pGEM3Zf(+). Dideoxy-nucleotide sequencing reactions were performed in both directions using the forward primer and the reverse primer (Pharmacia) with T7 DNA polymerase and [alpha-35S]dATP. The reactions were resolved on a 8M urea-5% polyacrylamide gel and visualized by exposing onto the X-ray film.
Results:
Two domains important for BCV neutralizing epitopes have been previously mapped to amino acid positions 324-403 and 517-720, respectively (4; see Fig. 1). The domain II overlaps with the probable BCV polymorphic region which have been suggested to reside in positions 456-592 (6). In order to further characterize the antigenic structure of BCV, a total of four mutant viruses resistant to neutralizing monoclonal antibody HB10-4 were selected. Neutralization-resistant mutants were confirmed by the lack of immunoreactivity of the S protein of the mutants with HB10-4 monoclonal antibody by immunoprecipitation (data not shown). To examine the nucleotide sequence responsible for the BCV neutralization, the region representing amino acids 351-721 was cloned from mutant viruses, and nucleotide sequence was determined (see Fig. 2). A wild-type BCV plaque was picked and carried in parallel with the mutant viruses in order to compare the sequences directly each other. By comparison of the nucleotide sequence of the HBm1 mutant virus with that of wild type BCV, we identified a single mutation of C to T at nucleotide position 1583 (see Fig. 3). The identical change was observed in 3 additional mutant viruses, HBm5, HBm9, and Hbm13. The single nucleotide change resulted in the change of alanine residue to valine residue at amino acid position 528. Our result indicates that the amino acid at position 528 on the S1 protein is directly related to formation of the antigenic structure responsible for the group A neutralizing epitope in BCV.
Discussion and Conclusions:
Using a series of BCV mutant viruses resistant to group A neutralizing monoclonal antibody HB10-4, we identified that a single nucleotide change in the S1 protein was responsible for the neutralizing epitope. This change was conserved in all four mutant viruses. It is interesting to note that the mutation occurred within the overlapping region of the polymorphic region and the domain II (see Fig. 4). This region appeared to be absent in mouse hepatitis virus strains A59 and JHM, but present in BCV. This is consistent with our finding that the BCV monoclonal antibody HB10-4 does not recognize the S proteins of A59 and JHM strain (data not shown) although they are in the same antigenic group. Our studies support the previous suggestion that the domains identified in the S1 portion and the polymorphic region are critical in the formation of the BCV antigenic structure.
References:
1. Lai, M. M. C. Microbiol. Rev. 56: 61. 1994.
2. Deregt, D., and Babiuk, L. A. Virology 161: 410. 1987.
3. Yoo, D. et al. Virology 179: 121. 1990.
4. Yoo, D. et al. Virology 183: 91. 1991.
5. Parker, S. et al. Virology 173: 664. 1989.
6. Parker, M. D. et al. J. Gen. Virol. 71: 263. 1990.
Comments:
Address questions and comments about this abstract to Dr. Dongwan Yoo ( dyoo@ovcnet.uoguelph.ca).
Previously presented at American Society for Virology on July 1996.