A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity

Abstract

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbs as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.

Fig. 1

Schematic diagrams of SARS-CoV S protein and RBD-Fc fusion protein. (A) There is a signal peptide (SP) located at the N-terminus of the S protein. The S1 domain contains a receptor-binding domain (RBD, residues 318–510) and the S2 domain contains two heptad repeat regions (HR1 and HR2) prior to transmembrane domain (TM). (B) Recombinant RBD-Fc molecule consists of RBD and a human IgG1-Fc fragment. R441A was generated by mutagenesis using the QuickChange XL kit. (C) Characterization of purified RBD-Fc and RBD-R441A mutant by SDS–PAGE analysis.

Fig. 2

Antibody responses in mice (A) and rabbits (B) immunized with RBD-Fc or RBD-R441A. Binding of serially diluted mouse or rabbits antisera collected after the third boost to the corresponding immunogen was measured by ELISA.

Fig. 3

Binding titers of mouse (A) and rabbit (B) antisera to the full-length S protein (FL-S) measured by ELISA.

Fig. 4

Neutralizing activity of mouse (A) and rabbit (B) antisera against SARS pseudoviruses. Infection of HEK293 cells expressing human ACE2 by SARS pseudoviruses (Tor2) was determined in the presence of mouse or rabbit antisera at a series of 3-fold dilutions. Percent neutralization was calculated for each sample and the average values were plotted.

Fig. 5

Reactivity of RBD-Fc and RBD-R441A mutant with anti-RBD mAbs that recognize different epitope conformations in RBD measured by ELISA. Antigens were coated to ELISA plates at 1 μg/ml and mAbs were tested at 10 μg/ml.

Fig. 6

Reactivity of RBD-Fc and RBD-R441A mutant with mouse anti-S antisera measured by ELISA. Antigens (FL-S, RBD-Fc, and RBD-R441A) were coated to ELISA plates at 1 μg/ml and mouse antisera were tested at 3-fold dilutions.

Fig. 7

Binding activity of RBD-Fc and RBD-R441A mutant to soluble ACE2. Soluble ACE2 was coated to plates at 2 μg/ml and binding of serially diluted RBD-Fc or R441A was measured by ELISA.

Fig. 8

Binding activity of RBD-Fc and RBD-R441A mutant to cell-associated ACE2. Binding of RBD-Fc and RBD-R441A mutant at 1 μg/ml to the HEK293 cells expressing human ACE2 was measured by flow cytometry.

Fig. 9

Inhibitory activity of RBD-Fc and RBD-R441A mutant against SARS pseudoviruses. Infection of HEK293 cells expressing human ACE2 by SARS pseudoviruses (Tor2) was determined in the presence of RBD-Fc or RBD-R441A at a series of 3-fold dilutions. Percent inhibition was calculated for each sample.