Amphotericin B treatment allows IAV fusion with IFITM3-containing endosomes.

(A) AmphoB rescues IAVpp fusion with IFITM3 expressing cells. IAVpp carrying BlaM-Vpr were used to infect A549 Vector, IFITM3, IFITM3-imNeonGreen (IFITM3-imNG), IFITM3-imTFP1, or 2M-IFITM3-imTFP1 cells in the presence of 1 μM AmphoB and viral fusion was measured by the BlaM assay. Data are fold-increase compared to fusion in the absence of AmphoB and SEM from 2 independent triplicate experiments. Lack of significant AmphoB effect on IAVpp fusion with unlabeled IFITM3 versus 2M-IFITM3-imTFP1 expressing cells is associated with their relatively high permissiveness for IAV fusion (see Fig 1F). (B) IAVpp carrying mCherry-2xCL-YFP-Vpr were imaged with A549-IFITM3-imTFP1 cells treated with 1 μM AmphoB. Time-lapse images show the viral particle entering an IFITM3+ compartment around 35 minutes and co-trafficking with IFITM3-imTFP1 until fusion occurs around 43 minutes. Scale bar 1.3 μm. (C) A fluorescence trace corresponding to the fusion event in panel C in the presence of AmphoB. (See S8 Movie). (D) Double-labeled IAVpp were pre-bound to 2M-IFITM3-imTFP1 cells as in (B). Images show IAVpp in an IFITM3+ cell that does not co-traffic with IFITM3+ compartments. Scale bar 5.9 μm. (E) Fluorescence traces for a single particle fusion in 2M-IFITM3-imTFP1 cells corresponding to the particle in panel E. (See S9 Movie). (F) Single IAVpp fusion efficiency in A459 Vector, IFITM3-imTFP1, 2M-IFITM3-imTFP1, and IFITM3-imTFP1 cells treated with 1 μM AmphoB; means are plotted with SEM from at least 11 independent experiments. IFITM3- and IFITM3+ denotes fusion with IFITM3-negative and -positive endosomes, respectively. (See also S5 Fig).