Fig 1. Arenavirus membrane permeability increases prior to virus fusion.

(A) Illustration of mCherry-CL-YFP-Vpr labeled single LASVpp fusion. LASVpp is internalized and trafficked to acidic endosomes where the viral membrane is permeabilized. Increases in viral membrane permeability lead to acidification of the virus’ interior which is manifested in YFP signal quenching. LASVpp-endosome fusion results in mCherry release into the cytoplasm and concomitant re-neutralization of the virus’ interior, seen as recovery of YFP signal. (B) Single LASVpp fusion with A549 cell. Time-lapse images (top) and fluorescence traces (bottom) show virus interior acidification (YFP quenching) at 12.3 min and fusion (YFP dequenching and mCherry loss) at 25.9 min (see S1 Movie). (C) A single MACVpp fusion event in A549 cell. Time-lapse images (top) and fluorescence traces (bottom) show virus interior acidification (YFP quenching) at 7.0 min and fusion (YFP dequenching and mCherry loss) at 29.0 min. (D) HIV-1 particles labeled with mCherry-CL-YFP-Vpr and pseudotyped with LASV, MACV GPc or IAV HA were attached to cells by spinoculation in the cold, and their entry/fusion was triggered by shifting to 37°C. Percentage of particles releasing mCherry is plotted. Data are means ± SD of 3 independent experiments. Results were analyzed by Student’s t-test. Numbers on the top of bars are numbers of total particles analyzed. Asterisks and NS on the top of bars represent the significance relative to the LASVpp fusion efficiency in A549 cells. **, p<0.01; NS, not significant. (E) Kinetics of single GPc and IAV pseudoviruse fusion with different target cells.