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.

We have previously documented unimpeded IAV lipid mixing activity in IFITM3expressing cells [30] . Analyses of lipid dye dequenching, irrespective of colocalization with IFITM3, did not reveal significant differences in the rate or extent of lipid mixing. This finding is in disagreement with the reduced IAV lipid dequenching in IFITM3-expressing cells reported in [39] . The reason for discrepant results is likely related to the use of a bulk lipid dequenching assay in [39] , as compared to the real-time single IAV tracking in our experiments. Importantly, in the present study, we were able to show the markedly slower rate of lipid redistribution to IFITM3-containing endosomes by focusing on events occurring in these compartments compared to lipid mixing in control cells. It should be noted that, in spite of exogenous incorporation of DiI into the viral membrane in the commonly used labeling protocol (e.g., [30, 67] ), the dye readily redistributes to both membrane leaflets, as we have demonstrated previously [68] . Thus, a slower lipid mixing between IAV and IFITM3-positive endosomes is consistent with a more restricted dye diffusion through the merged contacting leaflets of hemifused membranes, as compared to diffusion through both leaflets of a fusion pore.