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.

To assess the co-distribution of viruses with IFITM3 at the time of fusion in live cells, we generated fluorescently-tagged IFITM3 protein. Linear N-or C-terminal fusions of EGFP (or similar fluorophores) with IFITMs render IFITMs nonfunctional. However, we found that the coding sequence of EGFP inserted into the N-terminal region of IFITM3, predicted to reside in the cytoplasm [40] [41] [42] , generates a functional protein (Fig 1) . First, to verify correct subcellular distribution of the fluorescent construct, we co-expressed IFITM3-iEGFP (iEGFP stands for "internal" EGFP) with an N-terminally myc-tagged IFITM3 in HeLa cells and confirmed their colocalization by fixing and immunostaining the cells (Fig 1B) . Extensive colocalization between IFITM3-iEGFP and myc-IFITM3 suggests that subcellular localization of IFITM3 is not perturbed by incorporation of an EGFP tag. To test the functionality of the fluorescent construct, control A549 cells transduced with an empty vector (Vector) and cells transduced with IFITM3-iEGFP were infected with varied doses of influenza A/WSN/33 virus and the resulting infection measured by immunostaining cells for HA antigen. A549 cells were selected because they express very low endogenous levels of IFITM3 [15, 30, 31] . Cells expressing the EGFP-labeled IFITM3 were consistently much more resistant to influenza infection than control cells (Fig 1C) . Microscopic analysis revealed that cells expressing intermediate to high levels of IFITM3-iEGFP did not stain for HA antigen (Fig 1D) , demonstrating that IFITM3-iEGFP protects cells from IAV infection, similar to unlabeled IFITM3 [15, 30] .