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.

Live Cell Imaging Cells were plated onto chambered coverglass slides (Thermo Fisher Scientific) and cultured overnight. Before imaging, cells were incubated at 37 C for 1 hr with 5 mM of yellow/blue dextran (Life Technologies, 10,000 MW, L-22460) or 5 mM of Asante sodium green (TefLabs) in the presence or absence of 1 mM of (D) The COS7-vector, COS7-CAV1, or COS7-IFITM1 percent immobile fraction was calculated by first subtracting the normalized average of the 95 s postbleach images, then subtracting those values from 1 to determine the amount of membrane that did not recover. Results are representative of three independent experiments. (E) Schematic of IAV-induced cell-to-cell fusion assay. Individual cells (lower portion) exhibit distinct boundaries of actin (red lines) and clearly spaced nuclei (blue ovals). By comparison, after incubation with IAV and the addition of pH 5.0 buffer, the individual cells fuse and form syncytia, which possess multiple clustered nuclei with no intervening actin boundaries. (F) IAV-induced cell-to-cell fusion assay. Chilled IAV (PR8, moi 500-1,000) was incubated with the indicated stably transduced COS-7 cell lines from (A). Warm buffer of either pH 5.0 or 7.5 was added, followed by warm media. Cells were incubated for 5 hr and then fixed and stained to monitor fusion events for both actin (phalloidin, red), to highlight cellular boundaries (white dashed lines), and DNA (blue), to detect closely clustered nuclei present in syncytia. Scale bar, 10 mm. (G) Percentage of fused cells in experiments using pH 5.0 buffer in (F). Fusion events per 100 nuclei were calculated and values represent the average of three to five images from each of three independent experiments ± SD. (H) Immunoblot of the indicated cell lines from (A). The CIL antisera recognize an epitope that is identical in both IFITM1 and IFITM3. and survival (B) of WT and Ifitm3 À/À mice with or without intravenous administration of AmBisome (3 mg/kg) on days 0, 2, and 4 relative to intranasal inoculation with IAV X-31 (10,000 pfu); n > 3. (C) WT and Ifitm3 À/À mice were treated or not treated with AmBisome and then challenged with X-31 influenza as above. At day 6 postinfection, lung sections were prepared and stained with hematoxylin and eosin. (D) The indicated MEFs were incubated for 1 hr in the presence (red, +) or absence (blue, À) of 1 mM AmphoB and then challenged with X-31 or pandemic A/California/7/2009 (pH1N1) followed by fixation and immunostaining for NP. Numbers represent the mean percentage of infected cells of three separate experiments ± SD. (E) Immunoblot of the indicated cell lines from (D). (F) Model of IFITMs inhibiting viral fusion. Top panels depict HA-directed membrane fusion. Insertion of the viral fusion peptide (red) by the HA protein into the host's endosomal membrane is followed by an acid-induced conformational change in HA producing a hemifusion transition state (middle, arrows represent force (legend continued on next page)