Vemurafenib Inhibits Acute and Chronic Enterovirus Infection by Affecting Cellular Kinase Phosphatidylinositol 4-Kinase Type IIIβ

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  FIG 1. Vemurafenib inhibits enterovirus infection in a dose-dependent manner. (A) A549 cells were treated with different concentrations of vemurafenib before infecting the cells with EV1 and CVA9 (left) or different CVB serotypes, 1 to 6 (right). After 18 h postinfection, the cell viability was determined by crystal violet staining. The cell viability was normalized to control sample with no virus infection and 0 μM concentration of vemurafenib. The results are mean from two repeats with three technical replicates ± SEM (left) or three replicates ± SD (right). IC50 values were calculated using nonlinear regression analysis. (B) A549 cells were treated with different concentrations of vemurafenib, and the cytotoxicity was determined after short (6.5 h) and long time points (24 h) using CellTiter-Glo kit. The results are means from two repeats with three technical replicates ± SEM. CC50 values were calculated using nonlinear regression analysis, and selectivity indexes (SIs) for different viruses were determined by IC50 and CC50. (C) Different concentrations of vemurafenib were tested against poliovirus type 3 by treating A549 cells with the drug for 1 h and then infecting the cells with…

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  FIG 2. Vemurafenib affects both acute and chronic CVB4 infection. (A) Vemurafenib prevents the infection of CVB4 in pancreatic beta cell line in a dose-dependent manner. Mouse pancreatic beta cells, Min-6 cells, were treated with different concentrations of vemurafenib and infected or not with CVB4. The viability of cells was determined using crystal violet staining. The values are means ± SEMs from three replicates. (B) Vemurafenib decreases the levels of infectious CVB4 viral particles in the heart and pancreas tissue of mice. Hsd:ICR (CD-1) female mice at the age of 3 weeks were inoculated intraperitoneally with vemurafenib dissolved in DMSO and diluted in PBS (10 mg/kg) or with DMSO diluted in PBS once a day (starting on day 1) for 5 days. The animals were inoculated intraperitoneally with CV-B4 E2 (6 × 106 TCID50 in 200 μL PBS) on day 2. The animals were sacrificed on day 6, blood was collected, and portions of each organ (pancreas and heart) were frozen for determination of viral titer. Frozen organs were weighed, crushed using a TissueRuptor, homogenized in 0.5 mL of PBS, and then centrifuged at 2,000 × g for 10 min at 4°C. The supernatants were harvested to measure the titer of…

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  FIG 3. The inhibitory mechanism of vemurafenib is not through BRAF signaling. A549 cells were treated with different concentrations of a BRAF inhibitor, sorafenib (A), or Ras inhibitor, Salirasib (B), before EV1 or CVB3 infection. Also, noninfected control cells were included. After 18 h postinfection, the cell viability was determined by CTG assay. Values are means ± SEM from three replicates. (C) Confocal images of A549 cells infected with EV1 for 2 h with or without vemurafenib treatment compared to mock-infected cells. ERK is immunolabeled green. Scale bar, 40 μM.

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  FIG 4. Vemurafenib does not affect uncoating but prevents the translation and replication of EV1. (A) Direct effects of vemurafenib on EV1 particles was studied using a real-time spectroscopic assay, where the viral opening was monitored using SYBR green II, which intercalates with viral RNA, causing an increase in fluorescence. The opening was studied in storage buffer (2 mM MgCl2 in PBS) and opening ionic conditions (20 mM NaCl, 6 mM KH2PO4, 12 mM K2HPO4, and 0.01% faf-BSA). RNase was added to the reactions in order to separate the fluorescence that comes from the inside the particle (porous particle) from the fluorescence of the RNA outside the capsid. (B) Radioactively labeled EV1 particles were used to infect A549 cells. After 4 hpi, the cell lysates were treated with detergent, collected, and run in 5 to 20% sucrose gradients. The proportional amount of empty and intact particles was calculated by dividing the counts per minute of incident fractions with the total counts per minute value. The gradients are representative results of 3 three different experiments. (C) A549 cells were infected with EV1, after which vemurafenib was added at different times postinfection. The infection…

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  FIG 5. Vemurafenib does not prevent the actions of 2A and 3C proteases and 3D polymerase. (A) The effect of vemurafenib on viral proteases was studied by treating A549 cell homogenate with purified 2A and 3C proteases with or without the drug (5, 10, or 20 μM) being present. The results were detected by Western blotting, and known cellular targets for 2A and 3C, poly(A) binding protein (PABP), and stress granule assembly factor 1 (G3BP1), respectively, were immunolabeled. The results are representative of two separate experiments. (B) RNAs used in assays to study the effect of vemurafenib on viral 3D polymerase function. Asterisk indicates IRDye800 labeling site. (C) CVB3 3Dpol was incubated at room temperature for 15 min with RNA1 and various inhibitor concentrations or with 2.5% DMSO as a control. At 15 min, GTP and ATP were added along with RNA2, allowing the assembled 3Dpol-RNA1 complexes (≈60% of RNA1) to rapidly incorporate ATP and GTP and produce a +2 product, while RNA2 undergoes a slower reaction involving RNA binding followed by GTP incorporation to produce its +1 product. Top plot shows +2 and +1 product formation time courses for RNA1 and RNA2, respectively, and the bottom…

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  FIG 6. Vemurafenib affects PI4KB kinase and PI4P lipids. After overnight transfection with FAPP1-PH-GFP plasmid (A) or EGFP-HOSBP plasmid (B), HeLa cells were treated with vemurafenib (vemu), BF738735, OSW-1, or itraconazole (ITZ) for 1 h. Subsequently, the cells were fixed and stained with an antibody against PI4KIIIβ (PI4KB, red). Cell nuclei are visible in blue. (C) HeLa cells were infected with Renilla luciferase constructs RLuc-CVB3 WT, RLuc-CVB3-2C-AVIVAV, or RLuc-CVB3-3A-H57Y for 30 min (MOI of 0.1). Subsequently, the supernatant was replaced with DMEM containing the compound. Finally, the cells were lysed, and luciferase activity was measured or cell viability was determined at 7 hpi. Values are means ±SDs from three replicates, and IC50 values were calculated using nonlinear regression analysis. The change in the IC50 of H57Y mutant was statistically significant (P = 0.0001), while the change with AVIVAV mutant was not statistically significant (P = 0.09).