title: Mutations in or near the fusion peptide of the influenza virus hemagglutinin affect an antigenic site in the globular region.
abstract: We previously described a monoclonal antibody (Y8-10C2) that binds influenza virus hemagglutinin (HA) monomers but not native trimers. In this study, we demonstrated that Y8-10C2 binds to the globular domain of HA and found evidence that its epitope is located at the interface of adjacent subunits. We further showed that at elevated temperatures, the Y8-10C2 epitope is transiently exposed in trimers for antibody binding. Introduction of intrasubunit chemical cross-links into HA reversibly inhibited both Y8-10C2 binding to trimers at elevated temperatures and viral fusion activity, indicating that exposure of the epitope requires the normal conformational flexibility of the molecule. Prolonged incubation of Y8-10C2 with virus at an elevated temperature resulted in neutralization of viral infectivity, allowing selection of neutralization-resistant virus mutants. Mutants were divided into two classes based on a radioimmunoassay in which the virus is attached to polyvinyl: those with reduced affinity for Y8-10C2 or other monoclonal antibodies specific for the globular domain and those with no alteration in their interaction with Y8-10C2 or other antibodies. DNA sequencing of HA genes revealed that the first type of mutants possessed single amino acid substitutions in the Y8-10C2 epitope itself, while remarkably, the second type of mutants possessed single amino acid substitutions in or near the fusion peptide of the HA, which is located in the stem of the HA at a considerable distance from the Y8-10C2 epitope. These findings indicate that the conformational flexibility of the HA affects its antigenicity and that single amino acid substitutions in or near the fusion peptide influence the flexibility of the globular domains.
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
authors.forename: J W
authors.initials: JW
authors.lastname: Yewdell
authors.forename: A
authors.initials: A
authors.lastname: Taylor
authors.forename: A
authors.initials: A
authors.lastname: Yellen
authors.forename: A
authors.initials: A
authors.lastname: Caton
authors.forename: W
authors.initials: W
authors.lastname: Gerhard
authors.forename: T
authors.initials: T
authors.lastname: Bächi
mesh_terms: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D000956:Antigens, Viral / Q000235:genetics*; D000939:Epitopes / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016296:Mutagenesis; D009975:Orthomyxoviridae / Q000235:genetics*; D011487:Protein Conformation; D011489:Protein Denaturation; D011863:Radioimmunoassay; D014759:Viral Envelope Proteins / Q000235:genetics*; D014760:Viral Fusion Proteins / Q000235:genetics*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins