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

Wild type and EPS8-edited A549 cells were grown on coverslips and inoculated with WSN at a MOI of 5 in VGM with 0.25 μg/ml of TPCK-trypsin for 1 hour at 4°C. Warm VGM was added to the cells and infection progressed for the indicated length of time at 37°C. Cells were fixed with 4% paraformaldehyde in DPBS for 30 minutes at room temperature, permeabilized with 0.1% Triton-X in 0.1 M glycine for 5 minutes at room . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/592485 doi: bioRxiv preprint EPS8 facilitates uncoating of influenza A virus 12 Larson , Tran, et al. temperature, and blocked in 3% BSA in DPBS for a minimum of 30 minutes at room temperature. Cells were incubated sequentially with primary and secondary antibodies diluted in 3% BSA in DPBS: α-M1 (19 μg/ml) and chicken α-mouse AlexaFluor 594 (2 μg/ml); or α-RNP (1:1000) and donkey α-goat AlexaFluor 488 (2 μg/ml). Coverslips were mounted using mounting medium with 4',6-diamidino-2-phenylindole (DAPI) stain (Vector Laboratories, H-1200) and imaged using 20X and 40X objectives on an EVOS FL Auto (ThermoFisher). For M1 staining, a minimum of 100 M1-positive cells at 1.5 hpi were counted across 10 random fields of view for each condition in 2 separate biological replicates. Similar quantification was performed at 1 hpi, although fewer M1positive cells were present for all cell types. RNP localization was quantified by assessing a minimum of 100 cells across 10 random fields of view for each time point in each cell type across 3 separate biological replicates. Images were batch processed using ImageJ for quantification (Schneider et al., 2012) . Representative images for cytoplasmic and nuclear RNP staining were batch-processed separately to show staining distribution.