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.

Cell-to-Cell Fusion Assay Vector, IFITM1, or IFITM3 COS-7 cells and PR8 virus were chilled on ice before the addition of virus to cells (White et al., 1981 (White et al., , 1982 . Virus and cells were incubated at 4 C for 40 min. Cells were then rinsed and incubated with 37 C pH 5.0 or pH 7.5 buffer (10 mM of MES and 10 mM of HEPES in 13 PBS) for 5 min. Subsequently, the buffers were replaced with warm media and the cells incubated in a 37 C incubator for an additional 5 hr. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with Alexa Fluor 594-conjugated phalloidin (1:500, Life Technologies) and Hoechst 33342 (1:10,000, Invitrogen) before imaging. Fusion events were identified based on the formation of syncytia and nuclear aggregation. The percentage of fusion was calculated using a ratio of the nuclei within fused cells over the total nuclei for each microscopic field evaluated with more than five fields examined for each experimental condition.