Fig. 5: Effect of pretreatment, time of removal, and time of incubation on compound efficacy.

For all assays, cordycepin was used at 30 μM, BTZ-1 at 10 μM, and ADEP-42 at 10 μM. 229E was used to infect Huh-7 cells at MOI 2 unless otherwise indicated. Media samples harvested were analyzed for RNA release by RT-qPCR and for viral infectivity by TCID₅₀. A Shown are pretreatment assays with the assay timeline given in the schematic. Compounds were added 24 h before infection and removed at the time of infection. Media were harvested 24 hpi; RNA release (left) and viral infectivity (right) were then analyzed. For RNA release, mean ± SD are shown from n = 3 independent experiments, each performed in triplicates, ***p < 0.001. For viral infectivity, mean ± SD are shown from n = 1 experiment, performed in triplicate, **p < 0.01, *p < 0.05. B Shown are time of removal assays with assay timeline given in schematic. Compounds were added at 1 hpi and removed or not removed at 24 hpi as indicated. Media were harvested at 48 hpi; RNA release (left) and viral infectivity (right) were then analyzed. For RNA release, mean ± SD are shown from n = 2 independent experiments, each performed in triplicates, **p < 0.01, *p < 0.05. For viral infectivity, mean ± SD are shown from n = 1 independent experiment, performed in duplicate, **p < 0.01. C Shown are the time of incubation assays with the assay timeline given in the schematic. Cells were infected at MOI 0.02, compounds were added 1 hpi, and harvested at 24, 48, as well as 72 hpi. RNA release (left) and viral infectivity (right and panels below) were analyzed for each timepoint. For both RNA release and viral infectivity, mean ± SEM are shown from n = 3 independent experiments, each performed in triplicates, ****p < 0.0001, **p < 0.01, *p < 0.05.