Fluorescent IFITM3 does not restrict IAV lipid mixing.

(A) Schematic showing the single-virus hemifusion assay by monitoring dequenching of a lipophilic dye (SP-DiI18, colored green) resulting from lipid mixing between a virus and an endosome, yielding a green glow. HA was also fluorescently labeled (red), allowing it to be independently tracked. (B) Time series images show dequenching of SP-DiI18 upon entry of IAV co-labeled with AF647 (red) into A549 Vector cells. Scale bar 4 μm. (C) Fluorescence traces for the single IAV hemifusion event in (D) showing an increase in intensity of SP-DiI18 while the reference AF647 signal remains constant. The time for complete dequenching (Δt) and the dequenching ratio of final and initial mean intensities (If/Ii) are shown for this trace. (See S1 Movie.) (D) A549 cells stably transduced with IFITM3-imNG (IFITM3, blue) were monitored by time-lapse imaging, which shows SP-DiI18 dequenching as a result of lipid mixing between IAV and an endosome. Scale bar 17 μm. (See also S2 Fig) (E) Time series images show dequenching of SP-DiI18 upon entry of IAV co-labeled with AF647 with an endosome containing IFITM3-imTFP1. (F) Fluorescence traces for the single IAV lipid mixing event shown in (E). (See S3 Movie). (G) The average ratios of final (post-dequenching) and initial mean intensities (If/Ii) are shown from at least three independent experiments in A549 Vector and A549-IFITM3-imNG cells. Lipid mixing events were categorized based on whether they colocalized with IFITM3-imTFP1 puncta (IFITM3+) or occurred in the areas devoid of IFITM3-imNG signal (IFITM3-). Average ratios are plotted with standard errors. There are no significant differences in A549 Vector and IFITM3- average dequenching ratios or in A549 Vector and IFITM3+ endosomes. (H) The average dequenching times and standard errors are shown for A549 Vector, IFITM3- and IFITM3+ endosomes. The dequenching time between A549 Vector and IFITM3-negative endosomes was not significantly different, but lipid mixing was significantly slower in IFITM3+ endosomes.