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.

Nystatin, a pore-forming heptaen like AmphoB, was the only other compound found to rescue IAV infection from IFITM3. In contrast, the ionophores do not form pores but instead transport Na + by encasing the ion and shuttling it across the bilayer (Figure 3C) . This difference suggests that egress of a combination of ions (Cybulska et al., 1995; Hartsel et al., 1994) , or the pore itself may alter the membrane's properties and underlie AmphoB's negation of IFITM3. To investigate this, we used two small molecules, tetraethylammonium (TEA) and acetylcholine (ACh), to block AmphoB pores (Brutyan and McPhie, 1996; Terazima and Yoshino, 2010; Figure 3C ). We first determined conditions where TEA or ACh prevented the AmphoB-mediated efflux of Na + from endosomes ( Figure 3D ; quantitation provided in Figure S2C ). Importantly, we assume from these results that the diffusion of additional ions (i.e., K + and Cl À ) through the AmphoB pores is also blocked. In support of this, TEA also diminished the modest change in endosomal acidity observed with the addition of AmphoB (Figures S2A and S2D ). Next, using these conditions, we challenged the vector and IFITM3 cells with IAV and found that the blockade of AmphoB pores with either TEA or ACh had no effect on AmphoB's rescue of IAV replication ( Figure 3E ), arguing that alteration of endosomal ion concentration and pore conductance cannot account for either IFITM3mediated restriction or its negation by AmphoB.