Localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein.
To localize the epitopes recognized by monoclonal antibodies (MAbs) specific for the S1 subunit of the murine coronavirus JHMV spike protein, we have expressed S1 proteins with different deletions from the C terminus of S1. S1utt is composed of the entire 769-amino-acid (aa) S1 protein; S1NM, S1N, S1n(330), and S1n(220) are deletion mutants with 594, 453, 330, and 220 aa from the N terminus of the S1 protein. The expressed S1 deletion mutant proteins were examined for reactivities to a panel of MAbs. All MAbs classified in groups A and B, those reactive to most mouse hepatitis virus (MHV) strains and those specific for isolate JHMV, respectively, recognized S1N(330) and the larger S1 deletion mutants but failed to react with S1N(220). MAbs in group C, specific for the larger S protein of JHMV, reacted only with the S1utt protein without any deletion. These results indicated that the domain composed of the N-terminal 330 aa comprised the cluster of conformational epitopes recognized by MAbs in groups A and B. It was also shown that the epitopes of MAbs in group C were not restricted to the region missing in the smaller S protein. These results together with the fact that all MAbs in group B retained high neutralizing activity suggested the possibility that the N-terminal 330 aa are responsible for binding to the MHV-specific receptors. In investigate this possibility, we expressed the receptor protein and examined the binding of each S1 deletion mutant to the receptor. It was demonstrated that the S1N(330) protein as well as other S1 deletion mutants larger than S1N(330) bound to the receptor. These results indicated that a domain composed of 330 aa at the N terminus of the S1 protein is responsible for binding to the MHV-specific receptor.
Kubo
H
H
National Institute of Neuroscience, NCNP, Tokyo, Japan.
Yamada
Y K
YK
Taguchi
F
F
D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D003332:Coronaviridae / Q000276:immunology*; D000939:Epitopes; D008562:Membrane Glycoproteins / Q000276:immunology*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D015345:Oligonucleotide Probes / Q000737:chemistry; D011485:Protein Binding; D011991:Receptors, Virus / Q000378:metabolism*; D017384:Sequence Deletion; D064370:Spike Glycoprotein, Coronavirus; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes; D008562:Membrane Glycoproteins; D015345:Oligonucleotide Probes; D011991:Receptors, Virus; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus