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.

For co-localization analysis, the fixed cells were imaged on DeltaVision Elite (GE Healthcare) widefield microscope, using an Olympus PlanApoN 60x/1.42 NA oil immersion objective. Multiple Z-stacks with a spacing of 0.1 μm covering the entire thickness of the cells were acquired using a standard GFP/Cherry filter set. Deconvolution of the Z-stacks was done post acquisition to improve the signal-to-background ratio of the IFITM3-mNeonGreen vesicles and of mCherry labeled viruses using SoftWorX (DeltaVision, GE Healthcare). The deconvolved Z-stacks were used for quantitative volume based (voxel based) co-localization analysis using a custom protocol in the image analysis program Volocity (Perkin Elmer, Waltham, MA). Briefly, after background subtraction, the IFITM3 containing vesicles were identified as objects. mCherry virus particles with a size/volume threshold of 0.512 μm 3 (corresponding to a 2x2x2 voxel) and that were associated with IFITM3-expressing cells were identified. A virus particle was considered co-localized with IFITM3 when at least 50% of its volume overlapped within an IFITM3 expressing object. For every time point, at least 4 different fields of view containing multiple cells were imaged and the mean values of the % co-localization calculated.