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

Multicycle replication infections were performed by inoculating A549 cells at a multiplicity of infection (MOI) of 0.01 using virus diluted in virus growth media (VGM) (DMEM supplemented with penicillin/streptomycin [Corning cellgro], 25 mM HEPES [Corning cellgro], 0.3% BSA [Sigma-Aldrich]) with 0.25 μg/ml TPCK-trypsin. Supernatants were collected at indicated times and titered by plaque assay on MDCK cells (Matrosovich et al., 2006) or by a Nano-Glo viral titer assay by inoculating MDCK cells with reporter viruses and measuring luciferase activity (Karlsson et al., 2018; Tran et al., 2013) . Viral gene expression was measured by infecting cells with WSN PASTN viruses. Virus was diluted in VGM with 0.25-0.5 μg/ml TPCK-trypsin for A549 cells or Opti-MEM I (Invitrogen, 31985070) supplemented with 2% FBS for 293T and 293 cells. Viral gene expression was measured 8 hpi using a Nano-Glo luciferase assay kit (Promega). Viral attachment was quantified by inoculating A549 cells with bioluminescent PASN virions. Purified virus was diluted in VGM with 0.25 μg/ml TPCK-trypsin, applied to cells for 45 mins at 4°C, and removed. Cells were washed with cold VGM and bound virions were detected by performing a Nano-Glo assay.