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We have studied the kinetics of low-pH-induced fusion between erythrocyte membranes and membranes containing influenza virus hemagglutinin by using assays based on the fluorescence dequenching of the lipophilic dye octadecylrhodamine. Stopped-flow mixing and fast data acquisition have been used to monitor the early stages of influenza virus fusion. We have compared this with the kinetics observed for fusion of an NIH 3T3 cell line, transformed with bovine papillomavirus, which constitutively expresses influenza virus hemagglutinin (GP4f cells). Virus and GP4f cells both display a pH-dependent time lag before the onset of fluorescence dequenching, but of an order of magnitude difference, ca. 2 s versus ca. 20 s. We have adopted two strategies to investigate whether the difference in lag time reflects the surface density of acid-activated hemagglutinin, able to undergo productive conformational change. (i) Hemagglutinin expressed on the cell surface requires proteolytic cleavage with trypsin from an inactive HAO form; we have limited the extent of proteolysis. (ii) We have used infection of CV-1 cells with a recombinant simian virus 40 bearing the influenza virus hemagglutinin gene. The surface expression of hemagglutinin is a function of time postinfection. For low-pH-induced fusion of both types of cell with erythrocytes, the lag time decreases with increasing hemagglutinin densities. Our results do not indicate a cooperative phenomenon at the level of the principal rate-determining step. We also show in the instance of virus fusion, that the magnitude of the delay time is a function of the target membrane transbilayer lipid distribution. We conclude that for a given amount of pH-activated hemagglutinin per unit area of membrane, the kinetics of fusion is determined by nonspecific physical properties of the membranes involved.

Delay time for influenza virus hemagglutinin-induced membrane fusion depends on hemagglutinin surface density