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

. CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 sgRNA target Figure S2 . Validation of EPS8 editing in A549 cells. (A) Sequencing chromatograms for wild type and EPS8 knockout A549 cells. The sgRNA target site is underlined in black and the adjacent protospacer adjacent motif (PAM) in red. (B) ICE analysis of edited EPS8 clonal A549 cell lines. All inferred edits contributing at least 5% of the composite genotype are shown. The vertical dashed line indicates the cut site within the sgRNA target (bold letters). The gene exon sequence is shown in red. The two 3' nucleotides of the native splice donor site are underlined in black and potential alternative splice donors are underlined in blue. (C) Immunoblot of endogenous EPS8 and quantification of band intensity reveal low amounts of EPS8 protein in edited A549 clones despite mutations in the EPS8 locus that should create premature truncations. (D) The indicated deletions could result in low-frequency usage of alternative splice sites to generate a full length EPS8 protein. The wild type gene exon sequence is shown in red. The two 3' nucleotides of the native splice donor site are underlined in black and potential alternative splice donors are underlined in blue. Hypothetical alternative splice site usage that recreates the EPS8 open reading frame is shown for EPS8.1, where the alternative site matches the consensus splice donor sequence AU/GU, and EPS8.2, where the deletion repositions an alternative site AA/GU that is identical to the native site.