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 u sing a recently published comprehensive mass action kinetic model for HA-me diated fusion. This model includes the measurable steps in the fusion proce ss, i.e., first pore formation, lipid mixing, and content mixing of aqueous fluorescent markers. It contains two core parameters of the fusion site ar chitecture. The first is the minimum number of aggregated HAs needed to sus tain subsequent fusion intermediates. The second is the minimal number of t hose 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 f itted to obtain all best fits. Although each of the data sets required part icular parameter ranges for best fits, a consensus subset of these paramete r ranges could fit all of the data. Thus, this comprehensive model subsumes the available mass action kinetic data for the fusion of HA-expressing cel ls with erythrocytes, despite the differences in assays and experimental de sign, 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 fus ogenic aggregate, but they cannot undergo the essential conformational chan ge that initiates bilayer destabilization, thus solving a long-standing deb ate. Also, the similarity in rate constants for lipid mixing and content mi xing found here for HA-mediated fusion and by Lee and Lentz (Proc. Natl. Ac ad. Sci. U.S.A. 95:9274-9279, 1998) for PEG-induced fusion of phosphatidylc holine liposomes supports the idea that subsequent to stable fusion pore fo rmation, the evolution of fusion intermediates is determined more by the li pids than by the proteins.