Extended Data Fig. 5: Ganglioside supplementation of UGCG-KO cells.

a, Mean fold-change in viral RNA between 6 and 24 hrs in sgCtrl cells or UGCG-KO cells supplemented with 50 µM GD3 or vehicle (DMSO) for 24 hrs prior to infection with nHAV (left) or eHAV (right). Results are normalized to DMSO-treated cells at 6 hrs. n = 2 technical replicates. b, Mean NLuc activities in UGCG-KO2.1 cells supplemented with gangliosides (12.5-50μM) for 24 hrs prior to 18f/NLuc nHAV infection. sgCtrl cells were infected without supplementation. Cells were harvested for NLuc assay at 6 hpi. n = 3 technical replicates. c, Mean NLuc activity 18 hrs after nHAV infection of UGCG-KO1.3 cells supplemented with GM3, GD3 or GM2. n = 3 technical replicates. d, Mean relative NLuc activities ±s.d. in UGCG-KO2.1 cells supplemented with GM3 or GD3 (20-50 µM) 18 hrs after infection with nHAV (n = 5 independent experiments) or eHAV (n = 3 independent experiments). NLuc activities were normalized to GM3 supplementation (100 light units) in each experiment. Statistical comparison was by one-way ANOVA with Holm-Sidak’s multiple comparison test. e, Mean NLuc activity in Huh-7.5 cells 6 hrs after infection with nHAV pre-incubated with GM3, GM2 or GM1. n = 6 technical replicates. f, Immunoblot of ST8SIA1 (GD3 synthase, arrow) in lysates of polyclonal ST8SIA1-KO cells generated with different sgRNAs and sgCtrl cells. Actin included as a loading control. Data are from a single experiment. g, Mean NLuc activities in ST8SIA1-KO cells 6 and 24 hrs after 18f-NLuc nHAV infection. n = 4 technical replicates from one of 2 experiments with similar results. h, (left) Binding of eHAV to UGCG-KO1 vs. sgCtrl cells at 4 °C for 1 hr. Bars are means of n = 3 technical replicates. (right) Uptake of eHAV by UGCG-KO1 vs. sgCtrl cells following 2 hrs adsorption, removal of the inoculum, and 4 hrs additional incubation at 37 °C. Bars are means of n = 2 technical replicates i, Thermostability of 18f-NLuc nHAV following overnight incubation with 50 μM GD3 or DMSO at pH 5.5 and 4 °C, followed by additional incubation at the temperature indicated for 15 min prior to inoculation onto Huh-7.5 cells. Light units obtained with NLuc were normalized to light units from virus incubated for 15 min at 37 °C; lines reflect means of n = 2 technical replicates. All results shown as technical replicates were confirmed in independent experiments.

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