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. 2018 Oct 24;62(11):e01206-18. doi: 10.1128/AAC.01206-18

FIG 3.

FIG 3

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 ΔΔCT 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.