IFITM3 restriction of IAV fusion with A549 cells is not related to cholesterol accumulation in endosomes.

<p>(A) Sub-cellular distributions of cholesterol and IFITM3 in A549-Vector and A549-IFITM3 cells. Cholesterol and IFITM3 staining was done using filipin and anti-IFITM3 antibody, respectively. Images show confocal slices through the middle section of cells. (B) Filipin staining of A549 cells transduced with shRNA against NPC1 (upper panel) and of cells pretreated with 10 µM U18666A for 18 h (lower panel). (C) Intracellular filipin and IFITM3 signals are poorly correlated. Individual regions of interests within 91 cells were drawn to exclude plasma membrane fluorescence, followed by background subtraction and summation of fluorescence intensity within each region of interest. (D) Western blotting analysis of NPC1 expression in A549 cells transduced with scrambled shRNA (A549.shScr) or with shRNA specific to NPC1 (A549.shNPC1). Tubulin was used as a loading control. (E) IAVpp, VSVpp and EBOVpp fusion with A549.shScr and A549.shNPC1 cells measured by the BlaM assay. Data are means and SEM from 2 triplicate experiments (IAVpp and VSVpp) and 1 triplicate experiment (EBOVpp). (F) Single IAV lipid mixing activity in A549.shScr and A549.shNPC1 cells. Cells were allowed to bind AF488- and vDiD-labeled IAV in the cold and incubated at 37°C for 1 h. The number of vDiD dequenching events was normalized to the total number of cell-bound particles from two experiments (n>690 particles) for each cell line. Error bars are standard deviations. (G) Dose-dependence of U18666A effect on viral fusion. A549 cells were pre-incubated for 18 h with indicated concentrations of U18666A or DMSO (control). BlaM-Vpr-carrying pseudoviruses (MOI = 1) were allowed to fuse with cells for 90 min at 37°C in the presence of U18666A or DMSO. Data are means and SEM from 2 triplicate experiments. **, P = 0.005.