Gating kinetics of pH-activated membrane fusion of vesicular stomatitis virus with cells: stopped-flow measurements by dequenching of octadecylrhodamine fluorescence

Abstract

To identify the initial stages of membrane fusion induced by vesicular stomatitis virus, we performed stopped-flow kinetic measurement with fluorescently labeled virus attached to human erythrocyte ghosts that contained symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically using an assay based on mixing of the lipid fluorophore octadecylrhodamine. At 37 degrees C and pH values near the threshold for fusion, a lag phase of 2 s was observed. The lag time decreased steeply as the pH decreased, while the initial rate of fusion showed the reverse functional dependence on pH. The observed rapid fluorescence changes resulted from fusion of virus bound to the target, and the time lags were not due to association-dissociation reactions between virus and target. For a given pH value, the temperature dependence of the lag time was similar to that of the initial rate of fusion. The results were fitted to a multistate model similar to that resulting from ion channel gating kinetics. The model allows testing of hypotheses concerning the role of cooperativity and conformational changes in viral spike glycoprotein-mediated membrane fusion.