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.

Our data demonstrate that AmphoB increases IAV replication by overcoming IFITM3's protective effects in vitro. To determine the effects of AmphoB on IAV in vivo, we infected either WT or Ifitm3 knockout mice (Ifitm3 À/À ) with a low-pathogenicity strain of IAV (A/X-31 H3N2 [X-31]; (Everitt et al., 2012) . At the dose used, the X-31 strain produced a mild illness from which the WT mice made a full recovery ( Figures 6A and 6B ). In contrast, and consistent with our previous studies, the infected Ifitm3 À/À mice lost >25% of their body weight with signs of significant illness 6 days postinfection. Notably, WT littermates treated with AmBisome (3 mg/kg injected at days 0, 2, and 4 relative to viral inoculation) behaved identically to the Ifitm3 À/À mice, experiencing a clinical course usually observed with more pathogenic strains of IAV and manifest by severe symptoms in conjunction with a weight loss exceeding 25% of their starting values. We saw no signs of illness with AmBisome treatment alone in the absence of infection ( Figure S5 ). Evaluation of lung (C) Schematic diagram of AmphoB or nystatin transmembrane pores (left) permitting the passive diffusion of multiple monovalent cations (purple and red ovals) across the endosomal membrane (arrow indicates high to low concentration gradient). In comparison, the ionophores selectively bind Na + (red ovals) in a central region, effectively shielding the positive charge from the hydrophobic interior of the membrane and thus allowing the cation to be transported down the gradient. Adding TEA or ACh blocks the opening of the AmphoB pore, preventing ion transport (lower panel, gold oval). (D) Confocal images of the relative levels of endosomal sodium based on the fluorescent signal of ANG-2 (green) in A549-vector or A549-IFITM3 cells with or without treatment with AmphoB, TEA, or ACh. Quantitation provided in Figure S2C . Scale bar, 10 mm. (legend continued on next page) pathology at day 6 postinfection showed marked edema, pneumonia, and hemorrhage with substantial inflammation in the AmBisome-treated infected mice (either WT or Ifitm3 À/À ) as compared to the WT mice not exposed to AmBisome ( Figure 6C ). Indeed, in the setting of infection, there is little difference seen when comparing the AmBisome-treated WT lungs to ones from an Ifitm3 À/À animal.