Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus.
Oligonucleotide-directed mutagenesis of a cDNA encoding the hemagglutinin of influenza virus has been used to introduce single base changes into the sequence that codes for the conserved apolar "fusion peptide" at the amino-terminus of the HA2 subunit. The mutant sequences replaced the wild-type gene in SV40-HA recombinant virus vectors, and the altered HA proteins were expressed in simian cells. Three mutants have been constructed that introduce single, nonconservative amino acid changes in the fusion peptide, and three fusion phenotypes were observed: substitution of glutamic acid for the glycine residue at the amino-terminus of HA2 abolished all fusion activity; substitution of glutamic acid for the glycine residue at position 4 in HA2 raised the threshold pH and decreased the efficiency of fusion; and, finally, extension of the hydrophobic stretch by replacement of the glutamic acid at position 11 with glycine yielded a mutant protein that induced fusion of erythrocytes with cells with the same efficiency and pH profile as the wild-type protein. However, the ability of this mutant to induce polykaryon formation was greatly impaired. Nevertheless, all the mutant proteins underwent a pH-dependent conformational change and bound to liposomes. These results are discussed in terms of the mechanism of HA-induced membrane fusion.
D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004274:DNA, Recombinant; D005822:Genetic Vectors; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000235:genetics*; D007700:Kinetics; D050356:Lipid Metabolism; D008561:Membrane Fusion; D008970:Molecular Weight; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D009994:Osmolar Concentration; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000235:genetics / Q000502:physiology*; D014760:Viral Fusion Proteins
D004274:DNA, Recombinant; D006389:Hemagglutinins, Viral; D009838:Oligodeoxyribonucleotides; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins