Interferon-induced transmembrane protein 3 blocks fusion of sensitive but not resistant viruses by partitioning into virus-carrying endosomes

Suddala, Krishna C.; Lee, Christine C.; Meraner, Paul; Marin, Mariana; Markosyan, Ruben M.; Desai, Tanay M.; Cohen, Fredric S.; Brass, Abraham L.; Melikyan, Gregory B.

A549 IFITM3-imNG, IFITM3-imTFP1, or 2MIFITM3-imTFP1 cells were seeded onto 35 mm collagen-coated glass-bottom Petri dishes (MatTek, MA) one day prior to imaging and cultured in DMEM without phenol red supplemented with 10% FBS, penicillin, and streptomycin. Following a wash with room temperature PBS, cells were fixed in 3.5% paraformaldehyde in PBS for 10 min. For imaging with LysoTracker™, cells were seeded as above and incubated with 30 nM LysoTracker™ Red DND-99 (ThermoFisher) diluted in pre-warmed LCIB supplemented with 2% FBS for 30 min prior to fixation. Images were acquired on a DeltaVision microscope using an Olympus UPlanFluo 40x/1.3 NA oil immersion objective (Olympus, Japan). Multiple Z-stacks with a spacing of 0.1 μm covering the entire thickness of the cells were acquired and deconvolved.