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 next asked whether the presence of fluorescent IFITM3 affects the rate or extent of lipid exchange between IAV and the limiting membrane of a vesicle. The fold of SP-DiI 18 dequenching was measured by calculating mean ratios of single IAV SP-DiI 18 signals after and before dequenching (I f /I i ) in A549 Vector cells and in A549-IFITM3-imNG cells occurring within IFITM3+ punctae (as in Fig 2F) . The extent of SP-DiI 18 dequenching was independent of virus colocalization with IFITM3-imNG endosomes at the time of lipid mixing ( Fig 2G) . Since the extent of dequenching of lipophilic dyes is proportional to their fold-dilution (e.g., [47, 48] ), this result indicates that the average size of recipient endosomes is the same in control and IFITM3-expressing cells. However, the SP-DiI 18 dequenching time (Δt) was significantly longer in IFITM3+ endosomes compared to Vector cells (Fig 2H) , consistent with a hemifusion connection that is more restrictive for lipid diffusion in IFITM3+ endosomes incubating for 2 hr. Data are means and error bars are SEM from three independent experiments performed in triplicate (F) Expression levels of different IFITM3 constructs in transduced A549 cells. Stable cell lines expressing an empty Vector, unlabeled IFITM3, IFITM3-imNG,IFITM3-imTFP1, or 2M-IFITM3-imTFP1 were lysed and analyzed by Western blotting using rabbit anti-IFITM3 and mouse anti-Tubulin antibodies as a loading control. (See also S1 Fig for cell The average ratios of final (post-dequenching) and initial mean intensities (I f /I i ) are shown from at least three independent experiments in A549 Vector and A549-IFITM3-imNG cells. Lipid mixing events were categorized based on whether they colocalized with IFITM3-imTFP1 puncta (IFITM3+) or occurred in the areas devoid of IFITM3-imNG signal (IFITM3-). Average ratios are plotted with standard errors. There are no significant differences in A549 Vector and IFITM3-average dequenching ratios or in A549 Vector and IFITM3+ endosomes. (H) The average dequenching times and standard errors are shown for A549 Vector, IFITM3-and IFITM3+ endosomes. The dequenching time between A549 Vector and IFITM3-negative endosomes was not significantly different, but lipid mixing was significantly slower in IFITM3+ endosomes.