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 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 4ºC 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 37ºC. 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 4ºC, followed by additional incubation at the temperature indicated for 15 min prior to inoculation onto Huh-7.5 cells. NLuc normalized to virus incubated for 15 min at 37ºC; lines reflect means of n=2 technical replicates. All results shown as technical replicates were confirmed in independent experiments.