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.

IFITM3 blocks IAV fusion and prevents vRNPs from translocating to the nucleus (Feeley et al., 2011) . We examined the effect of AmphoB on IAV vRNP nuclear translocation (Figure 2A ). At the start of infection, the IAV nucleoprotein (NP) is associated with the vRNPs. By immunostaining for NP, we can follow vRNP distribution intracellularly. HeLa-vector or HeLa-IFITM3 cells were chilled on ice with IAV PR8. Next, the viral supernatant was (A) HeLa cells stably expressing either of two negative control short hairpin RNAs (shRNAs) against the firefly luciferase gene (shLuc-1 or shLuc-2) or shRNAs against IFITM3 (shIFITM3-1 and shIFITM3-2) were incubated for 1 hr in the presence (red) or absence (blue) of 1 mM AmphoB, then challenged with IAV WSN/33 (multiplicity of infection [moi] 0.2) or PR8 (moi 0.02) followed by immunostaining for HA. Numbers represent the mean percentage of infected cells of three separate experiments ± SD, as determined by imaging analysis software. (B) Whole cell lysates from the indicated cells in (A) were subjected to immunoblotting using the noted antibodies. kDa, kilodaltons. RAN serves as a loading control. (C) IFITM3's restriction of HA-mediated entry is overcome by AmphoB. A549 cells stably transduced with IFITM3 or with vector alone (Vector) were treated with 1 mM AmphoB (red) or buffer (blue) then challenged with the indicated pseudotyped MLV-GFP particles: WSN/33 (H1), PR8 (H1), Thai (H5), FPV (H7), or the g protein of VSV. Relative infection represents the percentage of GFP-positive cells normalized to that of A549-vector cells as determined by fluorescence-based imaging.