Studies of the membrane fusion activities of fusion peptide mutants of influenza virus hemagglutinin.
Influenza virus hemagglutinin (HA) fuses membranes at endosomal pH by a process which involves extrusion of the NH2-terminal region of HA2, the fusion peptide, from its buried location in the native trimer. We have examined the amino acid sequence requirements for a functional fusion peptide by determining the fusion capacities of site-specific mutant HAs expressed by using vaccinia virus recombinants and of synthetic peptide analogs of the mutant fusion peptides. The results indicate that for efficient fusion, alanine can to some extent substitute for the NH2-terminal glycine of the wild-type fusion peptide but that serine, histidine, leucine, isoleucine, or phenylalanine cannot. In addition, mutants containing shorter fusion peptides as a result of single amino acid deletions are inactive, as is a mutant containing an alanine instead of a glycine at HA2 residue 8. Substitution of the glycine at HA2 residue 4 with an alanine increases the pH of fusion, and valine-for-glutamate substitutions at HA2 residues 11 and 15 are without effect. We confirm previous reports on the need for specific HAo cleavage to generate functional HAs, and we show that both inappropriately cleaved HA and mutant HAs, irrespective of their fusion capacities, upon incubation at low pH undergo the structural transition required for fusion.
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D017354:Point Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013820:Thermolysin; D014162:Transfection; D014357:Trypsin
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014357:Trypsin; D013820:Thermolysin