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.

(E) Immunoblot of the indicated cell lines using the CIL antisera, which recognizes an epitope that is identical across all of the IFITM WT proteins and chimeras. Results are representative of three independent experiments. replaced with warm media (0 min). At the indicated times after warming, cells were evaluated for NP signal with a confocal microscope. vRNPs arrive in the nuclei by 90 min in the vector cells, with the NP signal increasing through to 120 min, and these events are inhibited by IFITM3 overexpression. Remarkably, when we treated the cell lines with AmphoB, both the vector and IFITM3 cells showed a strong increase in vRNP particles in their nuclei, although the vector cells were still more infected at 90 min. A time of addition experiment showed that AmphoB's effect was maximal when added 60 min prior to viral addition, consistent with inhibiting IFITM3 ( Figure S1E ). An immunoblot revealed that AmphoB treatment did not decrease IFITM3 levels ( Figure S1F ). Together with the pseudoparticle experiments, these data demonstrate that AmphoB's enhancement of IAV infection arises from it overcoming IFITM3's inhibition of viral fusion.