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

Biochem Biophys Res Commun. 2006 May 26;344(1):106-13. doi: 10.1016/j.bbrc.2006.03.139. Epub 2006 Mar 30.

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.

MeSH terms

  • Amino Acid Substitution / immunology*
  • Angiotensin-Converting Enzyme 2
  • Animals
  • Antibodies, Monoclonal / immunology
  • Antigens, Viral / genetics
  • Antigens, Viral / immunology
  • Arginine / genetics
  • Arginine / immunology*
  • Female
  • Immunoglobulin Fc Fragments / genetics
  • Immunoglobulin Fc Fragments / immunology
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / immunology*
  • Membrane Glycoproteins / metabolism*
  • Mice
  • Mice, Inbred BALB C
  • Peptidyl-Dipeptidase A / metabolism*
  • Point Mutation
  • Protein Structure, Tertiary / genetics
  • Rabbits
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • Spike Glycoprotein, Coronavirus
  • Viral Envelope Proteins / genetics
  • Viral Envelope Proteins / immunology*
  • Viral Envelope Proteins / metabolism*
  • Viral Vaccines / genetics*
  • Viral Vaccines / immunology

Substances

  • Antibodies, Monoclonal
  • Antigens, Viral
  • Immunoglobulin Fc Fragments
  • Membrane Glycoproteins
  • Recombinant Fusion Proteins
  • Spike Glycoprotein, Coronavirus
  • Viral Envelope Proteins
  • Viral Vaccines
  • spike glycoprotein, SARS-CoV
  • spike protein, mouse hepatitis virus
  • Arginine
  • Peptidyl-Dipeptidase A
  • ACE2 protein, human
  • Ace2 protein, mouse
  • Angiotensin-Converting Enzyme 2