Assessment of fusogenic properties of influenza virus hemagglutinin deacylated by site-directed mutagenesis and hydroxylamine treatment.
Influenza virus hemagglutinin (HA) subtype H7 expressed from a baculovirus vector in insect cells requires cysteine residues for palmitoylation. Mutant HA devoid of fatty acids shows hemagglutinating and hemolytic activities almost identical to those of the acylated wild-type HA (wt). Using a membrane mixing assay (R18), neither the kinetics nor the pH dependence of fusion induced by wt or mutant HA was significantly different from virus-induced fusion. HA-induced fusion of insect cells with human erythrocyte ghosts could also be demonstrated by a cytoplasmic content mixing assay. Both species of recombinant HA induced the flow of lucifer yellow from preloaded ghosts into the cytoplasm of HA-bearing cells. This indicates that membrane fusion mediated by wild-type and fatty-acid-free HA includes both leaflets of the lipid bilayers. Hydroxylamine treatment of wt HA (H7) and fatty-acid-free mutant HA present in lysates of insect cells led to the complete inhibition of hemolytic activity. Deacylation of spike proteins by NH2OH treatment of virus particles resulted in a block of hemolytic activity in influenza virus subtypes H7 and H10 as well as of that in the togaviruses Semliki Forest and Sindbis virus. However, the same treatment did not affect subtypes H2 and H3 or two vesicular stomatitis virus serotypes. With such a differential effect whether or not fatty acids are present in the spike proteins of the different virus particles, hydroxylamine must have other effects than just deacylation, and therefore seems unsuitable for the study of the biological functions of acylproteins.
Philipp
H C
HC
Institut für Immunologie und Molekularbiologie, Fachbereich Veterinärmedizin der Freien Universität Berlin.
Schroth
B
B
Veit
M
M
Krumbiegel
M
M
Herrmann
A
A
Schmidt
M F
MF
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D004910:Erythrocyte Membrane / Q000276:immunology / Q000502:physiology*; D006389:Hemagglutinins, Viral / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology*; D009980:Influenza A virus / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D006389:Hemagglutinins, Viral; D006898:Hydroxylamines; D011994:Recombinant Proteins; D019811:Hydroxylamine