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. Encapsulating such fluorescence probes in the lumen of target membranes permits specific detection of fusion events and exclusion of leakage without fusion. Using this assay, we show that influenza virus fuses with liposomes of different sizes with indistinguishable kinetics, suggesting that the starting curvature of such liposomes does not control the rate-limiting steps in influenza entry.