title: Single amino acid substitutions in the severe acute respiratory syndrome coronavirus spike glycoprotein determine viral entry and immunogenicity of a major neutralizing domain.
abstract: Neutralizing antibodies (NAbs) against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) spike (S) glycoprotein confer protection to animals experimentally infected with the pathogenic virus. We and others previously demonstrated that a major mechanism for neutralizing SARS-CoV was through blocking the interaction between the S glycoprotein and the cellular receptor angiotensin-converting enzyme 2 (ACE2). In this study, we used in vivo electroporation DNA immunization and a pseudovirus-based assay to functionally evaluate immunogenicity and viral entry. We characterized the neutralization and viral entry determinants within the ACE2-binding domain of the S glycoprotein. The deletion of a positively charged region Sdelta(422-463) abolished the capacity of the S glycoprotein to induce NAbs in mice vaccinated by in vivo DNA electroporation. Moreover, the Sdelta(422-463) pseudovirus was unable to infect HEK293T-ACE2 cells. To determine the specific residues that contribute to related phenotypes, we replaced eight basic amino acids with alanine. We found that a single amino acid substitution (R441A) in the full-length S DNA vaccine failed to induce NAbs and abolished viral entry when pseudoviruses were generated. However, another substitution (R453A) abolished viral entry while retaining the capacity for inducing NAbs. The difference between R441A and R453A suggests that the determinants for immunogenicity and viral entry may not be identical. Our findings provide direct evidence that these basic residues are essential for immunogenicity of the major neutralizing domain and for viral entry. Our data have implications for the rational design of vaccine and antiviral agents as well as for understanding viral tropism.
authors.affiliation: Aaron Diamond AIDS Research Center, The Rockefeller University, 455 1st Avenue, 7th Floor, New York, NY 10016, USA.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Yi
authors.forename: Lei
authors.initials: L
authors.lastname: Ba
authors.forename: Linqi
authors.initials: L
authors.lastname: Zhang
authors.forename: David D
authors.initials: DD
authors.lastname: Ho
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D005260:Female; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D017434:Protein Structure, Tertiary; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000276:immunology* / Q000472:pathogenicity / Q000502:physiology; D017384:Sequence Deletion; D064370:Spike Glycoprotein, Coronavirus; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000235:genetics; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000502:physiology; D014765:Viral Vaccines / Q000008:administration & dosage / Q000235:genetics; D014774:Virulence / Q000235:genetics / Q000276:immunology / Q000502:physiology
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D008562:Membrane Glycoproteins; D064370:Spike Glycoprotein, Coronavirus; D019444:Vaccines, DNA; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C578557:spike glycoprotein, SARS-CoV