Membrane fusion activity of influenza virus. Effects of gangliosides and negatively charged phospholipids in target liposomes

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Abstract
Fusion of influenza virus with liposomes composed of negatively charged phospholipids differs from fusion with biological membranes or zwitterionic liposomes with ganglioside receptors [Stegmann, T., Hoekstra, D., Scherphof, G., and Wilschut, J. (1986) J. Biol. Chem. 261, 10966.sbd.10969]. In this study, we investigated how the kinetics and extent of fusion of influenza virus, monitored with a fluorescence resonance energy-transfer assay, are influenced by the surface charge and the presence of receptors on liposomal membranes. The results were analyzed in terms of a mass action kinetic model, providing separate rate constants for the initial virus.sbd.liposome adhesion, or aggregation, and for the actual fusion reaction. Incorporation of increasing amounts of cardiolipin (CL) or phosphatidylserine (PS) into otherwise zwitterionic phosphatidylcholine (PC)/phosphatidylethanolamine (PE) vesicles results in a gradual shift of the pH threshold of fusion to neutral, relative to the pH threshold obtained with PC/PE vesicles containing the ganglioside GD1a, while also the rate of fusion increases. This indicates the emergence of a fusion mechanism not involving the well-documented conformational change in the viral hemagglutinin (HA). However, only with pure CL liposomes this nonphysiological fusion reaction dominates the overall fusion process; with pure PS or with zwitterionic vesicles containing CL or PS, the contribution of the nonphysiological fusion reaction is small. Accordingly, preincubation of the virus alone at low pH results in a rapid inactivation of the viral fusion capacity toward all liposome compositions studied, except pure CL liposomes. The results of the kinetic analyses show that with pure CL liposomes the rates of both virus .sbd. liposome adhesion and fusion are considerably higher than with all other liposome compositions studied. With pure PS vesicles or zwitterionic vesicles containing CL or PS, the rate of virus .sbd. liposome adhesion and the number of virus particles involved in the fusion process are higher than the corresponding parameters for PC/PE/GD1a liposomes, but the rate of the fusion reaction itself is the same. Incorporation of cholesterol in PC/PE/GD1a liposomes causes a relative enhancement of the rate of the fusion reaction itself, while relative to PC/PE liposomes the GD1a, serving as a receptor for the virus, only causes the rate of adhesion to increase. These results indicate that a specific interaction of the virus with a receptor on the target membrane does not influence the characteristics of the fusion reaction itself.