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

The putative enhancer with the strongest correlation score was EPS8, an adaptor protein involved in signaling via the epidermal growth factor receptor (EGFR) and other pathways as well as modulating of actin dynamics ( Figure 1D ) (Di Fiore and Scita, 2002; Hertzog et al., 2010) . To validate the results of the screen and confirm a pro-viral function for EPS8, we assessed the effect of EPS8 on viral gene expression and replication. EPS8 was transiently overexpressed in human embryonic kidney 293T cells and infected with a replication-competent reporter version of WSN (WSN PASTN) to quantitatively measure viral gene expression (Tran et al., 2013) . EPS8 overexpression increased viral gene expression during infection nearly two-fold relative to the empty vector control ( Figure 2A ). Endogenous and overexpressed EPS8 levels were confirmed by immunoblot. We then assayed viral titers when EPS8 was stably overexpressed in human lung epithelial A549 cells. Viral titers 24 hours postinfection (hpi) were increased by over 15 fold in stable EPS8-overexpressing cells relative to wild type (WT) cells ( Figure 2B ). Thus, overexpression of EPS8 enhances infection and replication in two different human cell lines, confirming the pro-viral correlation identified in the screen. We next used CRISPR-Cas9 to generate clonal EPS8 knockout A549 cells. Sanger sequencing confirmed genotypic changes predicted to result in knockout of EPS8 in two independent clonal lines (EPS8.1, EPS8.2) ( Figure S2A and S2B). Immunoblotting for endogenous EPS8 revealed a dramatic reduction in EPS8 protein levels but not a complete loss in our edited clones ( Figure S2C ).