Role of the fusion peptide sequence in initial stages of influenza hemagglutinin-induced cell fusion.
The fusion activity of influenza hemagglutinin (HA) and of HA proteins altered in the amino terminus of HA2 (fusion peptide) by site-directed mutagenesis (Gething, M.-J., Doms, R. W., York, D., and White, J. (1986) J. Cell Biol. 102, 11-23) was analyzed following expression in CV-1 cells using SV40-HA recombinant virus vectors. Fusion was monitored by the redistribution of lipid and cytoplasmic dyes between fluorescently labeled erythrocytes and HA-expressing CV-1 cells using spectrofluorometry and fluorescence microscopy. The kinetics of lipid redistribution after lowering the pH showed the same pattern for wild type HA and nonlethal mutants, although there were shifts in the pH threshold. The time for commitment to the fusogenic state and the temperature dependence of the processes leading to HA-mediated fusion were also the same for wild type and nonlethal mutants. However, striking differences were observed between wild type HA and the nonlethal mutants in their ability to induce pH-dependent redistribution from erythrocytes to HA-expressing cells of large molecular weight (M(r) > 10,000) fluorescently labeled dextran molecules. The data indicate that the kinetic processes which are measurable in the time range of seconds are insensitive to the structure of the fusion peptide. Surprisingly, however, the fusion peptide plays an important role in later processes related to pore widening which eventually results in delivery of the nucleocapsid into the cell.
Section on Membrane Structure and Function, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
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D000595:Amino Acid Sequence; D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D002522:Chlorocebus aethiops; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013539:Simian virus 40 / Q000235:genetics; D014759:Viral Envelope Proteins / Q000378:metabolism
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D014759:Viral Envelope Proteins