EPS8 facilitates uncoating of influenza A virus

Gloria P. Larson; Vy Tran; Shuiqìng Yú; Yíngyún Caì; Christina A. Higgins; Danielle M. Smith; Steven F. Baker; Sheli R. Radoshitzky; Jens H. Kuhn; Andrew Mehle

NCI-60 cell lines were seeded by groups of cell origin at 3 x 10 4 cells per well in 96-well plates (Greiner, 655948 for Operetta; Corning, 3604 for flow cytometry) and grown overnight in RPMI-1640 medium (Lonza, BE12-702F), supplemented with 10% heat-inactivated FBS (Sigma-Aldrich, 4135) at 37°C with 5% CO2. The cells were infected with WSN-GFP at MOIs 0.2 and 2. At 3 hours post-inoculation, the cells were washed with RPMI-1640 medium and fresh growth medium was added. WSN-GFP expression was measured by fluorescence microscopy 24 hours post-inoculation. Cells were fixed with 4% paraformaldehyde, and the nuclei were stained with Hoechst 33342 (Thermo Fisher, Catalog: H3570). WSN-GFP virus expression was detected by the Operetta-High Content Imaging System (PerkinElmer Inc.), and the percentage of GFP-positive cells were analyzed by Harmony4.1 software (PerkinElmer Inc.). WSN-GFP expression was evaluated by the flow cytometry (BD Biosciences, LSRFORTESSA) 24 hours post-inoculation. All infections were performed in triplicate, and two biological replicates performed for each MOI condition. Both approaches yielded similar results, and infectivity for each cell line was rank-ordered relative to MDCK cells. The relative infectivity of each cell line was log2-transformed and used as input for the COMPARE algorithm (Zaharevitz et al., 2002) .