Amphotericin B Increases Influenza A Virus Infection by Preventing IFITM3-Mediated Restriction

Lin, Tsai-Yu; Chin, Christopher R.; Everitt, Aaron R.; Clare, Simon; Perreira, Jill M.; Savidis, George; Aker, Aaron M.; John, Sinu P.; Sarlah, David; Carreira, Erick M.; Elledge, Stephen J.; Kellam, Paul; Brass, Abraham L.

The IFITM proteins can associate to form homo-and heteromeric complexes via their IM1 domains, and this interaction is required for restriction of IAV (John et al., 2013) . This prompted us to hypothesize that the multimerization of IFITM proteins inhibits viral fusion by altering the fluidity of the cell membrane. To test this idea, we measured the membrane fluidity of cells stably expressing IFITM1 using fluorescence recovery after photobleaching (FRAP). We chose to use COS-7 cells stably transduced with IFITM1 due to their planar morphology and because IFITM1 is predominantly cell-surface expressed as compared to IFITM3, which resides in late endosomes and lysosomes that were not amenable to FRAP studies due to their small size and continued movements (data not shown; Figure 5A ; Figure S4A ). Caveolin 1 (CAV1) was chosen as a control because it has a similar intramembrane topology to the IFITMs (Hoop et al., 2012; Yount et al., 2012) . After incubating the cells along with a fluorescent membrane probe (DiO), we then photobleached an area of the cell surface (white squares, Figure 5B ). We monitored the return of the DiO signal as readout for membrane fluidity by capturing serial images of the bleached areas ( Figure 5C) . These experiments showed that IFITM1 decreased membrane fluidity compared to vector cells, signified by the slower recovery and a greater than 2-fold increase in the immobile fraction in the IFITM1 cells ( Figure 5D ). Consistent with previous reports, and in contrast to IFITM1, CAV1 expression increased membrane fluidity, demonstrating the specificity of IFITM1's actions (Cai et al., 2004) . To test for a functional effect, we emulated elegant studies that established that IAV and other fusogenic viruses can induce tissue culture cells to form syncytia when exposed to low pH (White et al., 1981 (White et al., , 1982 . We incubated the COS-7 cell lines (vector, IFITM1, and IFITM3) with concentrated IAV PR8 on ice, followed by the addition of warm buffer at either pH 5.0 or 7.5. After replacing the buffer with media, we stained the cells for actin and DNA followed by image analysis. Quantitation of syncytia, based on clustering of nuclei and the absence of intervening bands of actin ( Figure 5E ), showed there were less fusion events occurring among the IFITM1 cells (1.5% ± 0.4% fused cells) as compared to either the IFITM3 cells or the vector control cells (3.6% ± 0.6% fused cells, p < 0.05; Figures 5F and 5G) . We interpret the lack of effect by IFITM3 in this assay to be attributable to its location in the late endosomes and lysosomes. The levels of the exogenously expressed IFITM1 and IFITM3 proteins were seen to be comparable by immunoblotting ( Figure 5H ). AmphoB did not alter either IFITM1's effect on membrane fluidity or its antifusion actions ( Figures S4B and S4C ), although this was not altogether unexpected given AmphoB's weak effect on IFITM1-mediated restriction. Therefore, we conclude that IFITM1 decreases membrane fluidity along with increasing resistance to IAV-induced cell-to-cell fusion.