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.

Results are the mean of three independent experiments ± SD. (D and E) A549-vector (blue) or A549-IFITM3 (red) cells were infected with increasing amounts of IAV (WSN/33, relative moi) in the presence or absence of AmphoB (D) or AmBisome (E). Infectivity was determined by immunostaining for surface HA protein. (A) HeLa-vector or HeLa-IFITM3 cells were incubated with AmphoB or buffer for 1 hr prior to being incubated on ice with IAV PR8. Warm media was added at time zero. Cells were fixed at the indicated time points, immunostained for the IAV NP protein (green) and stained for DNA, then analyzed by confocal microscopy. Image analysis software was used to define each cell's nuclear peripheries (blue lines based on the staining of nuclear DNA). Numbers represent the number of NP particles (green) present per nucleus at the indicated time points and are the mean ± SD of three independent experiments. Scale bar, 10 mM. (B) A549 cells stably transduced with vector alone (Vector) or with IFITM1 (M1), IFITM2 (M2), or IFITM3 (M3) were incubated for 1 hr with increasing concentrations of AmphoB followed by infection with IAV WSN/33 for 12 hr and immunostaining for HA. (C) Schematic diagram of the IFITM chimeras. The N-and C-terminal domains (NTD, CTD) and intramembrane domains 1 and 2 (IM1 and IM2) are shown, separated by the conserved intracellular loop (CIL). M1, IFITM1 (gray); M2, IFITM2 (green); M3, IFITM3 (red). The chimeric proteins (M3M1 and M1M3) are shown below, with the first M# representing the NTD's origins and the second M# denoting the consignation of the remaining portion of the protein.