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

Following attachment and entry, FLUAV traffics in an endosome that undergoes acidification which results in fusion of the endosomal and viral membranes. The function of EPS8 during endosomal acidification and fusion was tested using an acid bypass assay. Acid bypass replaces the canonical entry route with fusion of viral and plasma membranes at the cell surface, depositing vRNPs into the cytoplasm where subsequent steps of infection then proceed as usual (Banerjee et al., 2014; Matlin et al., 1981) . As in the attachment assay, virions bound to the surface of wild type or edited cells at 4°C to synchronize infection. Cells were shifted to 37°C and transiently held at acidic conditions (pH 5.0) to initiate fusion at the cell surface or held at physiological conditions (pH 7.4) permitting canonical entry to proceed as a control. EPS8 editing resulted in a decrease in viral gene expression when infections were initiated at physiological pH ( Figure 3E ), consistent with prior data showing defects in gene . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 5 Larson , Tran, et al. expression during unsynchronized infections ( Figure 2D and E). Bypassing canonical entry by treating cells with acidic conditions did not restore viral gene expression in the edited cells ( Figure 3E ), indicating EPS8 does not function during endosomal acidification. Together, these data establish that the effects of EPS8 during FLUAV infection are independent of virion attachment, endosomal entry, and HA-mediated fusion.