Variant influenza virus hemagglutinin that induces fusion at elevated pH.
The hemagglutinin (HA) glycoprotein of influenza virus performs two critical roles during infection: it binds virus to cell surface sialic acids, and under mildly acidic conditions it induces fusion of the virion with intracellular membranes, liberating the genome into the cytoplasm. The pH dependence of fusion varies for different influenza virus strains. Here we report the isolation and characterization of a naturally occurring variant of the X31 strain that fuses at a pH 0.2 units higher than the parent strain does and that is less sensitive to the effects of ammonium chloride, a compound known to elevate endosomal pH. The bromelain-solubilized ectodomain of the variant HA displayed a corresponding shift in the pH at which it changed conformation and bound to liposomes. Cloning and sequencing of the variant HA gene revealed amino acid substitutions at three positions in the polypeptide. Two substitutions were in antigenic determinants in the globular region of HA1, and the third occurred in HA2 near the base of the molecule. By using chimeric HA molecules expressed in CV-1 cells from simian virus 40-based vectors, we demonstrated that the change in HA2 was solely responsible for the altered fusion phenotype. This substitution, asparagine for aspartic acid at position 132, disrupted a highly conserved interchain salt bridge between adjacent HA2 subunits. The apparent role of this residue in stabilizing the HA trimer is consistent with the idea that the trimer dissociates at low pH. Furthermore, the results demonstrate that influenza virus populations contain fusion variants, raising the possibility that such variants may play a role in the evolution of the virus.
D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006389:Hemagglutinins, Viral / Q000235:genetics; D006461:Hemolysis; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000235:genetics; D008081:Liposomes; D008561:Membrane Fusion; D008854:Microscopy, Electron; D009154:Mutation; D013539:Simian virus 40 / Q000235:genetics; D013329:Structure-Activity Relationship; D014779:Virus Replication / Q000187:drug effects
D006389:Hemagglutinins, Viral; D008081:Liposomes; D000643:Ammonium Chloride