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.

To investigate our observations regarding AmphoB and IFITM3, we first evaluated the effect of AmphoB on IAV replication in IFITM-depleted cells. HeLa cervical adenocarcinoma cells were stably transduced with either of two short hairpin RNAs (shRNAs) targeting IFITM3 or two negative control shRNAs against firefly luciferase ( Figures 1A and 1B) . HeLa cells were chosen because they express relatively high levels of endogenous IFITM3 and differing endogenous levels of IFITM3 strongly correlate with AmphoB's effects on IAV replication (Feeley et al., 2011; Roethl et al., 2011) . The cell lines were then challenged with either of two strains of IAV, H1N1 A/WSN/33 (WSN/33) or H1N1 A/PR/38 (PR8), with or without AmphoB. The loss of IFITM3 in untreated cells increased the infection of both viruses, with the enhancement being greater for PR8 (25-fold) than for WSN/33 (5-fold). In the IFITM3-depleted cells treated with AmphoB, we saw no increased infection with WSN/33 and a modest additional increase in replication with PR8 (1.7-and 2.4-fold, respectively), suggesting that the majority of AmphoB's enhancement of IAV infection is due to overcoming IFITM3. The IFITMs act on the viral envelope protein-dependent stage of entry by blocking fusion. Therefore, we tested the effect of AmphoB on the infectivity of viral pseudoparticles that express the HA1, HA5, or HA7 proteins. Consistent with its overcoming IFITM3, AmphoB treatment enhanced the infectivity of all of the HA-expressing particles ( Figure 1C) . Notably, the infectivity of particles bearing PR8's HA1 was uniquely increased in the vector cells treated with AmphoB, suggesting that the residual infectivity seen when IAV PR8 is used with IFITM3-depleted cells ( Figure 1A ) is the result of an intrinsic property of the PR8 HA1 envelope. Interestingly, while IFITM3 did modestly inhibit VSV-g pseudoparticle infection, this block was not alleviated by AmphoB.