FIG 5.

Vandetanib and spautin-1 inhibit dengue maturation and egress in a VPS34-independent manner. Comparison of (A) extracellular and (B) intracellular viral titer at 24 hpi during treatment at the IC₉₀ doses of spautin-1 (2.4 μM) or vandetanib (3.6 μM) revealed abundant intracellular virus despite reduced extracellular virus in the presence of each compound. Cells were pretreated for 1 h, infected for 1 h at an MOI of 0.5 PFU/cell, and posttreated for an additional 23 h. N = 4, one-way ANOVA with Dunnett’s multiple-comparison test. ns, not significant (P > 0.05); **, P < 0.01; ****, P < 0.0001. (C) Huh7 cells transfected with a luciferase-expressing RNA replicon were treated with 3 μM vandetanib (orange) or 1% DMSO vehicle control (black) for 1 h prior to transfection and up to 52 h posttransfection. Luciferase intensity was quantified as a measure of viral protein abundance. Luciferase intensity was shown as relative light units (RLU). The use of an RNA replicon that is directly transfected into cells reflects only translation and RNA replication steps of viral infection, not viral packaging or cell exit. N = 4, two-way ANOVA with Sikak’s multiple-comparison test. Statistical significance was defined as P < 0.05; no significant differences were observed between DMSO and vandetanib treatments at any time point. (D) VPS34 was not required for spautin-1 or vandetanib anti-dengue activity. Wild-type and VPS34 knockout Huh7 cells were treated with 5 μM spautin-1, 5 μM vandetanib, or DMSO. Cells were pretreated, infected at an MOI of 0.5, and posttreated as described above for panels A and B. At 24 hpi, cellular supernatant was collected and titered by plaque assay. N ≥ 3. One-way ANOVA with Dunnett’s multiple-comparison test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.