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 EPS8 locus was edited in A549 cells by lentiviral expression of CRISPR/Cas9 components. Vesicular stomatitis Indiana virus (VSIV) glycoprotein G-pseudotyped lentivirus was generated by transfecting 293T cells with the plasmids psPAX2, pMD2.G, and pLentiCRISPR (Addgene 52961, (Sanjana et al., 2014) ) modified to encode a single-guide RNA (sgRNA) targeting EPS8 (5'-TCAACTTACTTCATCTGAGA-3', Supplemental Figure 2 ). A549 cells were transduced with this virus, placed under puromycin selection (0.5 μg/ml), and single cells were cloned. Pooled 293 cells edited at the EPS8 locus were created by Synthego by transfecting cells with Cas9 RNPs containing an sgRNA targeting exon 5 (5'-GCACTTGACTACCTTTGTCC-3') (Supplemental Figure 3) . 293 cells were single cell cloned. Edited alleles in both cell types were identified by PCR amplification of the locus, Sanger sequencing of the products, and inference of CRISPR edits (ICE) analysis (Hsiau et al., 2019) (Supplemental Figures 2A-B and 3A-B) . Knockouts predicted by ICE analysis were assessed by immunoblot. Stable expression of EPS8 in cells was achieved by lentivirus gene delivery. The gene delivery vector pLX304-EPS8 was created by Gateway recombination of pENTR223-EPS8 (DNASU HsCD00505776; (Seiler et al., 2014) ) into pLX304 (Addgene 25890) and encodes the 822 amino acid splice variant (NCBI XP_024304650). Virus was produced by transfecting 293T cells with plasmids pLX304-EPS8, psPAX2, and pMD2.G. Wild type and EPS8-edited A549 and 293 cells were transduced with this virus and selected with blasticidin to obtain cells stably expressing EPS8.