Fusion of membrane vesicles bearing only the influenza hemagglutinin with erythrocytes, living cultured cells, and liposomes.
Membrane vesicles, bearing only the influenza viral hemagglutinin glycoprotein, were reconstituted following solubilization of intact virions with Triton X-100. The viral hemagglutinin glycoprotein was separated from the neuraminidase glycoprotein by agarose sulfanilic acid column. The hemagglutinin glycoprotein obtained was homogenous in gel electrophoresis and devoid of any neuraminidase activity. A quantitative determination revealed that the hemolytic activity of the hemagglutinin vesicles was comparable to that of intact virions. Incubation of fluorescently labeled hemagglutinin vesicles with human erythrocyte ghosts (HEG) or with liposomes composed of phosphatidylcholine/cholesterol or phosphatidylcholine/cholesterol/gangliosides, at pH 5.0 but not at pH 7.4, resulted in fluorescence dequenching. Very little, if any, fluorescence dequenching was observed upon incubation of fluorescently labeled HA vesicles with neuraminidase or glutaraldehyde-treated HEG or with liposomes composed only of phosphatidylcholine. Hemagglutinin vesicles were rendered non-hemolytic by treatment with NH2OH or glutaraldehyde or by incubation at 85 degrees C or low pH. No fluorescence dequenching was observed following incubation of non-hemolytic hemagglutinin vesicles with HEG or liposomes. These results clearly suggest that the fluorescence dequenching observed is due to fusion between the hemagglutinin vesicles and the recipient membranes. Incubation of hemagglutinin vesicles with living cultured cells, i.e. mouse lymphoma S-49 cells, at pH 5.0 as well as at pH 7.4, also resulted in fluorescence dequenching. The fluorescence dequenching observed at pH 7.4 was inhibited by lysosomotropic agents (methylamine and ammonium chloride) as well as by EDTA and NaN3, indicating that it is due to fusion of hemagglutinin vesicles taken into the cells by endocytosis.
Lapidot
M
M
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Nussbaum
O
O
Loyter
A
A
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D004910:Erythrocyte Membrane / Q000502:physiology* / Q000648:ultrastructure; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000302:isolation & purification; D006461:Hemolysis; D006801:Humans; D009980:Influenza A virus / Q000502:physiology; D007700:Kinetics; D008081:Liposomes; D008223:Lymphoma; D051379:Mice
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D008081:Liposomes