Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

Spence, Jennifer S.; Krause, Tyler B.; Mittler, Eva; Jangra, Rohit K.; Chandran, Kartik

The lipophilic dye DiD spontaneously incorporates into membranes and displays fluorescence self-quenching at high concentrations. Lipid mixing between viral and host bilayers, as in hemifusion or fusion, enables lateral diffusion of the dye accompanied by dequenching or increased fluorescence intensity (31, 32) . Virions were labeled so that the fluorescence signal of self-quenched DiD was detectable prior to dequenching. DiD dequenching resulted in a sharp increase in signal, which we define here as at least a doubling of DiD intensity within 20 s, and a net increase in intensity of 2-to 5-fold within 1 min was typically observed ( Fig. 1B and C; see also Movie S1 in the supplemental material). In our experiments, increases in DiD signal sorted strictly into one of two groups: very fast or very slow. Fast dequenching, as indicated by a representative trace (Fig. 1D , solid line), occurred irrespective of nucleocapsid release (data not shown) and appears to indicate specifically triggered lipid mixing events. We attribute the significantly lower rate of DiD dequenching (Fig. 1D , dotted line) to nonspecific transfer of the dye to host membranes, possibly through particle degradation. Only fast events constituted "dequenching" in this study. We found that the extent of dequenching varied somewhat among DiD-labeled particles. This may reflect differences in the volumes of endocytic compartments, as well as in the concentration of dye within the viral membrane, as some heterogeneity in self-quenching was noted. Dequenching events occurring at later postinfection time points generally exhibited greater amplitude between the quenched and dequenched states (data not shown).