FIG 3.

DHBTs inhibit DENV2 infection at an early stage in the life cycle. (A) HEK293 cells were incubated with DENV2 at an MOI of 3 FFU/cell for 1 h. Uninternalized virus was removed by an acid wash, and cells were refed with a normal growth medium. At the indicated times postattachment, the medium was removed and replaced with a medium containing DMSO or 5 μM SKI-417616. Supernatants were collected at 48 h p.i., and titers were determined on Vero cells. (B) HEK293 cells were infected with DENV2 at an MOI of 3 FFU/cell in the presence of DMSO or 5 μM SKI-417616. Total RNA was collected at 24 h postinfection, and genome equivalents were detected by RT-qPCR using primer and probe sets specific for the 3′ UTR (dark shaded bars) or E (light shaded bars) region of the viral genome. (C) Cells were treated with carrageenan (4 μg/ml), chlorpromazine (20 μM), or SKI-417616 (10 μM) and were then infected for 1 h with DENV2 (MOI, 20 FFU/cell). External virus was removed and infectious centers quantified as described in Materials and Methods. ***, P < 0.001. (D) Cells were treated with 20 μM chlorpromazine, 10 μM SKI-417616, or DMSO (control), followed by incubation with fluorescently labeled transferrin. After 1 h, cells were fixed, and transferrin internalization was analyzed by confocal microscopy. (E) BHK21-DVrep cells were treated with 2-fold-increasing concentrations of ribavirin (3.75 μM to 60 μM) or SKI-417616 (612.5 nM to 10 μM). Cells were lysed at 48 h posttreatment, and firefly luciferase activity was measured by luminescence. Values were normalized to those for DMSO-treated controls.