Measuring single-virus fusion kinetics using an assay for nucleic acid exposure

The kinetics by which individual enveloped viruses fuse with membranes provide an important window into viral-entry mechanisms. We have developed a real-time assay using fluorescent probes for single-virus genome exposure than can report on stages of viral entry including or subsequent to fusion pore formation and prior to viral genome trafficking. We accomplish this using oxazole yellow nucleic-acid-binding dyes, which can be encapsulated in the lumen of target membranes to permit specific detection of fusion events. Since increased fluorescence of the dye occurs only when it encounters viral genome via a fusion pore and binds, this assay excludes content leakage without fusion. Using this assay, we show that influenza virus fuses with liposomes of different sizes with indistinguishable kinetics by both testing liposomes extruded through pores of different radii and showing that the fusion kinetics of individual liposomes are uncorrelated with the size of the liposome. These results suggest that the starting curvature of such liposomes does not control the rate-limiting steps in influenza entry.