IFITM3-mediated restriction of IAV fusion is not related to cholesterol accumulation in endosomes of MDCK cells.

<p>(A) Sub-cellular distributions of cholesterol (filipin staining) and IFITM3 (antibody staining) in MDCK-Vector and MDCK-IFITM3 cells. Images show confocal sections through the middle of cells. (B) Filipin staining of MDCK cells pretreated with 20 µM U18666A for 18 h or mock-treated cells. (C) Dose-dependence of U18666A effect on viral fusion. MDCK-Vector cells were pretreated 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. (D–F) pH distributions in IAV-carrying endosomes of MDCK cells measured using AF488- and CypHer5E-labeled viruses. Viruses were pre-bound to cells in the cold and incubated at 37°C for 45 min before acquiring images. Calculated pH values are shown for MDCK cells without (D) and with pretreatment with 20 µM U18666A for 18 h (E), as well as for MDCK-IFITM3 cells (F). Data are from 10 image fields each.