Fig 4. Single LASVpp fusion with DF-1 cells.

(A) Illustration of fusion of mCherry-2xCL-YFP-Vpr labeled single LASVpp in an acidic endosome. An increase in the virus membrane permeability leads to quenching of the intraviral YFP signal (green) in acidic environment. Subsequent virus fusion with the endosomal membrane results in a loss of mCherry signal (red) through a fusion pore and concomitant re-neutralization of virus interior, as evidenced by YFP signal dequenching. (B) LASVpp fusion events (YFP dequenching) with instant mCherry release (quick fusion pore dilation). Time lapse images (left), fluorescence traces (middle top), instant velocity (middle bottom) and trajectory (right) of single LASVpp fusion with DF-1-LAMP1-WT cell showing YFP quenching at 31.3 min and YFP dequenching/mCherry loss at 34.7 min corresponding to virus interior acidification and fusion, respectively (see S1 Movie). (C) LASVpp fusion events with delayed mCherry release relative to YFP dequenching. Time lapse images (left), fluorescence traces (middle top), instant velocity (middle bottom) and trajectory (right) of single LASVpp fusion with a DF-1-LAMP1-WT cell showing YFP quenching at 39.7 min, YFP dequenching at 42.7 min and mCherry loss at 43.2 min (arrows), indicating virus interior acidification, small fusion pore formation and fusion pore dilation to a diameter exceeding the size of mCherry, respectively (see S2 Movie). (D) LASVpp fusion events (YFP dequenching) without mCherry release. Time lapse images (left), fluorescence traces (middle top), instant velocity (middle bottom) and trajectory (right) of single LASVpp fusion with a DF-1-LAMP1-WT cell showing YFP quenching at 42.6 min and dequenching at 47.4 min without mCherry loss (see S3 Movie).