Amphotericin B Increases Influenza A Virus Infection by Preventing IFITM3-Mediated Restriction

Lin, Tsai-Yu; Chin, Christopher R.; Everitt, Aaron R.; Clare, Simon; Perreira, Jill M.; Savidis, George; Aker, Aaron M.; John, Sinu P.; Sarlah, David; Carreira, Erick M.; Elledge, Stephen J.; Kellam, Paul; Brass, Abraham L.

Intrigued by recent reports, we tested the role of VAPA, cholesterol, and oleic acid in IFITM3-mediated restriction. Overexpression of VAPA modestly decreased IFITM3-mediated restriction of IAV in our assays. Based on our use of the same (F) Whole cell lysates from the indicated cells in (E) were subjected to immunoblotting using the indicated antibodies. Actin serves as a loading control. (G) Niemann-Pick type c1 (NPC) or wild-type (WT) primary human fibroblasts infected with the indicated IAVs with or without AmphoB (Ampho) treatment. (H) Confocal images of primary human fibroblasts in (G) stained with filipin (white) to detect cholesterol and immunostained for LAMP1 (red) to identify the late endosomal and lysosomal compartments. Scale bar = 15 mM. Figure S4A ). Nuclear DNA was stained with DAPI (blue). Scale bar, 10 mM. (B) Fluorescence recovery after photobleaching of the plasma membrane. COS7-vector or COS7-IFITM1 cells were stained with DiO and the unbound dye removed. Cells were photobleached in the indicated area (white square) with a 488 laser, and a series of pictures out to 95 s postbleach were taken. Images are shown for prebleach, postbleach, and 17 s postbleach (t 1/2 of IFITM1 recovery). (C) COS7-vector, COS7-CAV1, or COS7-IFITM1 cell fluorescence recovery was quantified using Leica Lite software, with each individual time point representing the average of ten normalized readouts. PRISM software was then used to plot the best-fit exponential decay line. Results are representative of three independent experiments.