Fig 1: K22 inhibits ZIKV replication and acts at a postentry stage. Vero cells were treated for 4 h with different concentrations of K22. Cells were infected with ZIKV (MOI = 0.1 TCID50/cell) for 1 h before the medium was removed and medium containing the different concentrations of K22 was added. (a) At 24 h and 48 h p.i., cells were harvested and E protein expression was analyzed by flow cytometry. (b) Viral infectivity was determined by harvesting the supernatant of cells and performing a TCID50 assay on Vero cells. (c) Cells treated with the compound were stained with a live/dead cell marker, and the MFI was determined by flow cytometric analyses. Shaded bars represent the positive control of dead cells. (d) Commercially available cell toxicity assays (cytotoxicity; CytoTox 96 nonradioactive cytotoxicity assay; Promega) as well as viability assays (proliferation; MultiTox-Fluor multiplex cytotoxicity assay; Promega) were performed to determine the cytotoxic effect of K22 on Vero B4 cells at 24 h posttreatment. (e) To evaluate the direct effect of K22 on viral stability, ZIKV in DMEM was incubated for 2 h at room temperature with the different K22 concentrations, and the viral titer was determined as the number of TCID50 per milliliter. (f) Time-of-addition assays were performed to analyze the effect of K22 on the early virus-cell interaction. K22 at various concentrations was added at specific time points relative to the time of infection with ZIKV, and the cell culture supernatants were collected at 24 h p.i. for measurement of virus release. The results represent the mean (bar) + SD (n = 2 or 3). Dotted lines indicate the limit of detection. n.d., not detected. P values were determined by 2-way ANOVA, followed by Dunnett's multiple-comparison test. ****, P = 0.0001; ***, P = 0.001; **, P = 0.01; *, P = 0.05; n.s., not significant.
Fig 2: K22 inhibits the replication and infectivity of hepacivirus and pestivirus. Huh7-Lunet-N-hCD81-FLuc cells were pretreated with K22 for 3 h before supernatant removal and infection with JcR-2a virus for 3 h at 37°C. After this, the inoculum was removed and the compounds were added and left until the end of the experiment. (a) At 48 h p.i., the cells were lysed and Renilla luciferase (replication) activity was measured. (b) Infectious supernatants were harvested and transferred onto target Huh7-Lunet-N-hCD81-FLuc cells. Target cells were lysed at 72 h p.i., and Renilla luciferase activity (infectivity) was determined. Data are depicted as relative light units (RLU). (c) The viability of Huh7-Lunet-N-hCD81-FLuc cells after K22 treatment was determined upon MTT assay, and the data were normalized to those for untreated cells. Depicted are the data normalized to those for untreated cells. (d) The viability of BT cells after K22 treatment was determined using commercially available cell toxicity assays (cytotoxicity; CytoTox 96 nonradioactive cytotoxicity assay; Promega) as well as viability assays (proliferation; MultiTox-Fluor multiplex cytotoxicity assay; Promega). Depicted are the data normalized to those for the DMSO-treated cells. (e to h) BT cells were treated with K22 for 4 h before supernatant removal and infection with BVDV SuwaCp (e, f) or BVDV SuwaNcp (g, h) for 1 h at 37°C before the medium was removed and medium containing the different concentrations of K22 was added. (e, g) Cells were lysed at 48 h p.i., and intracellular RNA was extracted. qRT-PCR was performed, and viral replication was calculated using the ??CT method and is depicted as the fold induction compared to that for DMSO-treated control cells. (f, h) Viral titers were determined by serial dilution on BT cells at 4 days p.i. The results represent the mean (bar) + SD (n = 3). The dashed lines indicate the limit of detection. P values were determined by 1-way ANOVA followed by Dunnett's multiple-comparison test. ****, P = 0.0001; ***, P = 0.001; **, P = 0.01; *, P = 0.05; n.s., not significant.
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