Comprehensive kinetic analysis of influenza hemagglutinin-mediated membrane fusion: role of sialate binding

The data of Danieli et al. (J. Cell Biol. 133:559-569, 1996) and Blumenthal et al. (J. Cell Biol. 135:63-71, 1996) for fusion between hemagglutinin (HA)-expressing cells and fluorescently labeled erythrocytes has been analyzed using a recently published comprehensive mass action kinetic model for HA-mediated fusion. This model includes the measurable steps in the fusion process, i.e., first pore formation, lipid mixing, and content mixing of aqueous fluorescent markers. It contains two core parameters of the fusion site architecture. The first is the minimum number of aggregated HAs needed to sustain subsequent fusion intermediates. The second is the minimal number of those HAs within the fusogenic aggregate that must undergo a slow "essential" conformational change needed to initiate bilayer destabilization. Because the kinetic model has several parameters, each data set was exhaustively fitted to obtain all best fits. Although each of the data sets required particular parameter ranges for best fits, a consensus subset of these parameter ranges could fit all of the data. Thus, this comprehensive model subsumes the available mass action kinetic data for the fusion of HA-expressing cells with erythrocytes, despite the differences in assays and experimental design, which necessitated transforming fluorescence dequenching intensities to equivalent cumulative waiting time distributions. We find that HAs bound to sialates on glycophorin can participate in fusion as members of the fusogenic aggregate, but they cannot undergo the essential conformational change that initiates bilayer destabilization, thus solving a long-standing debate. Also, the similarity in rate constants for lipid mixing and content mixing found here for HA-mediated fusion and by Lee and Lentz (Proc. Natl. Acad. Sci. U.S.A. 95:9274-9279, 1998) for PEG-induced fusion of phosphatidylcholine liposomes supports the idea that subsequent to stable fusion pore formation, the evolution of fusion intermediates is determined more by the lipids than by the proteins.