Kinetics and extent of fusion between Sendai virus and erythrocyte ghosts: application of a mass action kinetic model

Abstract

The kinetics and extent of fusion between Sendai virus and erythrocyte ghosts were investigated with an assay for lipid mixing based on the relief of self-quenching of fluorescence. The results were analyzed in terms of a mass action kinetic model, which views the overall fusion reaction as a sequence of a second-order process of virus-cell adhesion followed by the first-order fusion reaction itself. The fluorescence development during the course of the fusion process was calculated by numerical integration, employing separate rate constants for the adhesion step and for the subsequent fusion reaction. Dissociation of virus particles from the cells was found to be of minor importance when fusion was initiated by mixing the particles at 37.degree. C. However, besides the initiation of fusion, extensive dissociation does occur after a preincubation of a concentrated suspension of particles at 4.degree.C followed by a transfer of the sample to 37.degree. C. The conclusion drawn from the levels of fluorescence increase obtained after 20 h of incubation is that in principle most virus particles can fuse with the ghosts at 37.degree. C and pH 7.4. However, the number of Sendai virus particles that actually fuse with a single ghost is limited to 100-200, despite the fact that more than 1000 particles can bind to one cell. This finding may imply that 100-200 specific fusion sites for Sendai virus exist on the erythrocyte membrane. A simple equation can yield predictions for the final levels of fluorescence for a wide range of ratios of virus particles to ghosts. The rate constants of adhesion were very high, close to those expected in diffusion-controlled processes. The fusion rate constants were relatively small, but the calculations indicate that 1-2 min after the onset of the interaction between virus particles and ghosts at 37.degree.C and pH 7.4 most of the virus particles associated with the ghost have fused, provided that the number of virus particles per cell is below 100.