FIG 3.

Niclosamide augments ST-148 virucidal activity and functions most potently early in the dengue infectious cycle. (A) Chemical structures and (B) ROCS overlay of ST-148 and niclosamide. (C) Dose-response curves for ST-148 and niclosamide treatment of DENV2-infected Huh7 cells revealed IC₅₀ values of 0.38 μM and 0.28 μM, respectively. Compound addition, viral growth, and cytotoxicity assays were performed as described in the legend to Fig. 2C. (D) Virucidal activity of compounds at the concentrations indicated was measured after 60-min incubation of drugs with virus-containing solutions at 37°C. Viral titer was quantified by plaque assay on BHK-21 cells. n = 3. Statistical significance was evaluated by one-way ANOVA with Sidak’s multiple-comparison test and indicated as follows: *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant. (E) Infected Huh7 cells were treated with ST-148 and niclosamide at the IC₉₀ doses for different durations, as indicated, within a single cycle of infection (24 h). On the left, cells were pretreated for 2 h prior to infection, compounds were removed during a 1-h infection at an MOI of 0.5 PFU/cell and then readministered after removal of viral inoculum. On the right, cells were similarly infected and compounds were first introduced 4 h after starting the infection. Cell supernatant was collected at 24 h postinfection (hpi), and infectious virus was quantified by plaque assay. n = 4, one-way ANOVAs with Dunnett’s multiple-comparison tests, ns, not significant (P > 0.05); ***, P < 0.001. (F) To test the effect of ST-148 and niclosamide on viral RNA replication, Huh7 cells were infected for 1 h at an MOI of 0.5 PFU/cell. Infections were allowed to proceed for 4 h to allow viral entry and the initiation of infection, after which compounds were administered at the IC₉₀ doses of 5.6 μM ST-148 or 0.66 μM niclosamide. Intracellular RNA was collected at the time points indicated and quantified by qRT-PCR. Only the control for inhibited vRNA replication, 50 μM MK0608 treatment, significantly reduced vRNA load relative to the 1% DMSO vehicle control (P < 0.001 at 18 hpi, P < 0.0001 at 24 hpi). N = 4, two-way ANOVA with Dunnett’s multiple-comparison test. (G) When administered at an increased dose, ST-148 and niclosamide were effective during later, postentry stages. After 4 h of infection, 10 μM ST-148 or 1 μM niclosamide was added; extracellular virus was collected at 24 hpi and quantified by plaque assay. N = 3, one-way ANOVA with Dunnett’s multiple-comparison test, ****, P < 0.0001.