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 determine the specificity of AmphoB's actions, we compared its effect on A549 cells stably expressing IFITM1, IFITM2, or IFITM3. These experiments showed that while AmphoB effectively counteracts IFITM2 and IFITM3, its neutralization of IFITM1 was considerably less, with only a 1.4-fold increase in infection observed ( Figure 2B ). Next, chimeric IFITM proteins were constructed to find what regions of IFITM3 confer AmphoB sensitivity (John et al., 2013 ). IFITM3's N-terminal domain (NTD) was fused with the remaining portions of IFITM1 to generate M3M1, and the NTD of IFITM1 was fused to IFITM3 to produce M1M3 ( Figure 2C ). The M3M1 chimera was less affected by AmphoB treatment similar to wild-type (WT) IFITM1, and the M1M3 protein was rendered ineffective by AmphoB (Figure 2D ). The levels of the WT IFITM proteins in the cell lines used for these experiments were purposefully matched to the expression levels of the chimeras, resulting in less restriction than seen with higher-expressing cell lines ( Figure 2E ). These results reveal that AmphoB impacts IFITM2 and IFITM3 greater than IFITM1 and that this susceptibility resides in amino acids 51-133 of IFITM3.