Figure 3.

Gangliosides bind the HAV capsid and mediate a post-endocytosis step in viral entry. a. Mean percent nHAV bound to UGCG-KO1.3 and -KO2.1 cells versus sgCtrl cells following 1 hr incubation at 4ºC. n=3 technical replicates. b. Mean percent nHAV internalized by UGCG-KO versus sgCtrl cells at 37ºC. Cells were incubated with nHAV for 1 hr, washed with PBS, and reincubated for 5 hrs prior to lysis. n=3 technical replicates. c. nHAV binding to (left) GD1a and (right) GD3 gangliosides immobilized on plastic strips after overnight incubation at pH 4.0–8.0. Bound nHAV was detected with R10 antibody²¹. Bars show mean absorbance of n=2 technical replicates (GD1a) or in n=2 independent experiments, each with 2 technical replicates (GD3). Dotted lines indicate mean background absorbance without virus. d. Trypsin treatment inhibits nHAV entry. Cells were pretreated with trypsin for 30 min at 37ºC, and harvested 6 hpi for NLuc assay. Bars represent means from two independent experiments. e. Post-adsorption/endocytosis rescue of nHAV replication in UGCG-KO1.3 cells by (left) GD1a and (right) GD3. Cells were inoculated with 18f-NLuc nHAV at 37ºC, then placed in suspension by trypsin treatment, washed extensively, and re-plated prior to supplementation with 20–50 μM gangliosides or LacCer. Cells were assayed for NLuc activity 24 hrs later. Bars are means of n=3 technical replicates. f. HAV eclipse assay. UGCG-KO1.3 and sgCtrl cells inoculated with 18f-NLuc nHAV for 1 hr at 4ºC were shifted to 37ºC (time=0) and assayed subsequently at intervals for intracellular virus that had not uncoated by measuring NLuc activity in Huh-7.5 indicator cells 6 hpi. Neutralization with R10 antibody confirmed NLuc was generated by infectious virus. Data are means ± s.e.m. from 3 independent experiments (2 including R10 neutralization), each with 2–3 technical replicates. At 8 and 12 hrs, p=0.0192 and 0.0208 for UGCG-KO1.3 versus sgCtrl, respectively, by two-way ANOVA with Sidak’s test for multiple comparisons. g. Thermostability of 18f-NLuc nHAV incubated with 2 μM GD1a or DMSO for 1 hr at pH 5.5 at 37ºC, then re-incubated at 35–59ºC for 15 min prior to inoculation of Huh-7.5 cells. Results were normalized to virus incubated at 35ºC; n=3 technical replicates.