Extended Data Fig. 3: Characterization of clonal UGCG-KO cell lines generated with two different sgRNAs.
From: Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus
a, Schematic showing exons (boxed segments, translated region deeply shaded) and introns (horizontal lines) in the UGCG gene. Chromatograms obtained by Sanger sequencing of the indicated regions of UGCG in the Huh-7.5 sgCtrl and two UGCG-knockout clones, KO1.3 (exon 1) and KO2.1 (exon 2). The locations of indels disrupting the open reading frame and resulting in truncated protein expression are highlighted in red. The AUG codon in the first exon at which translation originates is highlighted in green. b, Detection of GM1 ganglioside on the surface of sgCtrl, UGCG-KO1.3 and UGCG-KO2.1 cells by flow cytometry using Alexa-488 fluorophore conjugated cholera toxin (CtB). Isotype indicates parallel staining with an Alexa-488 conjugated anti-mouse secondary antibody. Absence of detectable GM1 ganglioside on the surface confirmed both clones are functional knockouts of ganglioside synthesis. Data representative of two independent experiments. c, Laser-scanning confocal images of HAV antigen (K24F2 mAb, green) in sgCtrl and UGCG-KO1.3 cells 5 days after inoculation with naked (nHAV) and quasi-enveloped (eHAV) HM175/18f virus confirm a lack of replication in the knockout cells. Images shown are representative of 3 random fields in one experiment. Scale bar = 10 μm. d, Mean human rhinovirus B14 (RV-B14) RNA abundance measured by real-time RT-PCR in sgCtrl and UGCG knockout cells at the indicated times post infection. n = 2 technical replicates. e, Mean hepatitis C virus (HCV) RNA abundance measured by real-time RT-PCR (normalized to actin mRNA) in sgCtrl and UGCG knockout cells at 16 and 64 hrs after infection with JFH1 virus. n = 2 technical replicates.
