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. Author manuscript; available in PMC: 2017 Apr 21.
Published in final edited form as: Cell Chem Biol. 2016 Apr 21;23(4):443–452. doi: 10.1016/j.chembiol.2016.03.010

Figure 1. GNF-2’s inhibition of DENV2 is mediated by cellular Abl kinases and an additional target not shared with imatinib.

Figure 1

(A) Structures of Abl kinase inhibitors GNF-2 and imatinib. (B–E) Infections were performed by incubating cells with DENV2 NGC inoculum for one hour at 37 degrees, after which the cells were washed to remove non-adsorbed virus. Unless otherwise indicated, all infections were performed at a multiplicity of infection (MOI) of 1. To quantify antiviral effects, culture supernatants were harvested at 24 hours post-infection (hpi), corresponding to a single-round of infection, and the yield of infectious viral particles was quantified by viral plaque formation assay. (B) GNF-2 exhibits greater antiviral potency than imatinib in dose-response experiments quantifying the effect of inhibitor when present during the entirety of a single round of DENV2 NGC infection. (C) Time-of-addition experiments were performed by infecting BHK21 cells at MOI of 1 and varying the time of inhibitor treatment. For “PRE” conditions, either cells or viral inoculum were preincubated with imatinib or GNF-2. For “CO” conditions, inhibitors were present during the one hour incubation of viral inoculum with cells. For “POST” conditions, inhibitors were added following the initial one-hour infection and washes. Final inhibitor concentration was 15 μM for all conditions. Both GNF-2 and imatinib inhibit DENV2 when added at 0-5 hours post-infection, but only GNF-2 exhibits antiviral activity when preincubated with the DENV2 inoculum prior to infection. (D) Cells were reverse-transfected with siRNA Smartpool™ targeting c-Abl or with siGlo control. 48 hours later, cells were infected; viral yields were determined 72 hpi. (E) Wildtype 3T3 cells or murine embryo fibroblast (MEF) cells genetically deficient for c-Abl were infected with DENV2 NGC at MOI of 1 or 10; viral yields were measured at 72 hpi. The absence of c-Abl due to RNAi or genetic knockout is associated with reduced DENV2 replication. Each experiment was performed n ≥ 2, and viral plaque formation assays were performed in triplicate. Representative data are shown for the mean ± stdev of the viral plaque formation assays for a representative experiment.