A. Huh7 cells were electroporated with a DV2 reporter replicon and treated with inhibitors at 48 hours post-electroporation. Luciferase activity was quantified at 72 h post-electroporation and is plotted as a percentage of the DMSO-treated samples. Mean values and standard deviation from n=3 independent experiments are shown.
B. Huh7 cells were transfected with a DV2(GVD) reporter replicon, and immediately treated with compounds. Luciferase activity was quantified at 6 hours post-transfection and is plotted as a percentage of the DMSO-treated samples. Mean values and standard deviation from n=3 independent experiments are shown.
C. The assay described in panel B was used to determine IC50 of each small molecule against the DV2(GVD) replicon. Cells were treated with a range of small molecules concentrations, and the concentrations that lead to 50% inhibition in firefly luciferase signal (IC50) were calculated using the nonlinear fit variable slope model (GraphPad Software). IC90 values obtained against the infectious virus in Table 1 and Figure S1 were plotted versus the IC50 values for inhibition of viral translation in the DV2(GVD) replicon assay, and the resulting curve was fit by linear regression to illustrate correlation of antiviral potency and inhibition of translation.
D. Huh7 cells were mock infected or infected with DV2 (MOI of 10) for 1 hour and then were treated with DMSO or 2 µM QL-XII-47. Cover slips were collected at 24 hours post-infection and stained for the DV2 C (in green) and NS5 (in red) proteins. Nuclei were stained with DAPI (in blue). The images were taken at 400x magnification, and representative images from n>3 independent experiments are shown.
E. Huh7 cells were mock-infected or infected with DV2 (MOI of 1) for 1 hour, and then treated with inhibitors at a final concentration of 2 µM. At 24 hours post- infection, the cell lysates were collected and analyzed for the presence of DV2 E and NS5 proteins, as well as for the presence of GAPDH by Western blotting. A representative experiment out of n=2 repeats is shown.