Fig 1.
ILK knockdown reduces DENV infection.
(A) Representative western blots of phosphorylated (p-) and indicated proteins in the mock- (M) and DENV serotype 2 (DENV2)-infected control (shLuc) and ILK knockdown (shILK) A549 cells at indicated hours post-infection (hpi) are shown. The ratios of phosphorylated to total Akt relative to mock-infected control cells are shown. (B) Representative western blots of indicated proteins in A549 cells infected without (-) or with (+) DENV2 and treated with indicated concentrations of OSU-T315 at 24 hpi are shown. (C) Representative images of DENV2-infected A549 cells stained with anti-dsRNA antibody (green) at 24 hpi are shown. Nuclei were counterstained with DAPI. (D) The viral yields in indicated control and ILK knockdown A549 cells infected with indicated serotypes of DENV at 24 hpi are shown. Data represent means + SD (error bars) in panel D. *p < 0.05, **p < 0.01.
Fig 2.
ILK knockdown reduces DENV infection after virus binding and entry.
(A and B) The luciferase activity (A) and representative western blots of indicated proteins (B) in Huh7 cells with DENV replicon expressing luciferase and control shRNA (shLacZ) or shRNA targeting ILK 3’UTR (shILK-1) or CDS (shILK-2) are shown. (C and D) The luciferase activity (C) and cell viability (D) of Huh7 replicon cells treated with indicated concentrations of OSU-T315 or 0.05% Triton X-100 for 24 hours are shown. The viability of cells without OSU-T315 treatment was set as 100%. (E) Representative western blots of indicated proteins in Huh7 cells infected without (-) or with (+) DENV and treated with the indicated concentration of OSU-T315 for 24 hours are shown. The ratios of viral protein to β-actin relative to infected control (shLacZ) cells or infected cells without OSU-T315 treatment are shown. Data represent mean + SD (error bars) in bar graphs. *p < 0.05, **p < 0.01, ***p < 0.001. RLU, relative light unit. BD, below detection.
Fig 3.
ILK attenuates IFN-induced STAT1/2 activation and ISG expression.
(A-B) The protein levels of IFN-β (A) and mRNA levels of MxA (B) in control (shLuc) or ILK knockdown (shILK) A549 cells infected without (-) or with (+) DENV at 24 hours post-infection (hpi) are shown. The MxA level in control cells without DENV infection was set as 1. Data represent mean + SD (error bars). *p < 0.05, **p < 0.01, ***p < 0.001. (C) Representative western blots of phosphorylated (p-) and indicated proteins in mock- (M) and DENV-infected control or ILK knockdown A549 cells at indicated hpi are shown. The ratios of phosphorylated to total STAT1 and STAT2 relative to infected control cells at 24 hpi are shown. (D) Representative western blots of indicated proteins in A549 cells infected without (-) or with (+) DENV and treated with the indicated concentration of OSU-T315 for 24 hours are shown. The ratios of MxA and NS1 to β-actin as well as phosphorylated to total STAT1 relative to infected cells without OSU-T315 treatment are shown.
Fig 4.
ILK regulates SOCS3 expression to attenuate IFN signaling.
(A) Representative western blots of the indicated proteins in mock- (M) and DENV-infected control (shLuc) or ILK knockdown (shILK) A549 cells at indicated hours post-infection (hpi) are shown. The ratios of SOCS1 or SOCS3 to β-actin relative to mock-infected control cells are shown. (B-C) Representative RT-PCR images of the indicated genes (B) and western blots of phosphorylated (p-) and indicated proteins (C) in control and SOCS3 knockdown (shSOCS3) A549 cells infected with (+) or without (-) DENV at indicated or 24 hpi are shown. The ratios of phosphorylated to total STAT1 and STAT2 relative to infected control cells are shown. (D) The viral yields in indicated A549 cells at 24 hpi are shown. Data represent mean + SD (error bars). *p < 0.05. (E) Representative western blots of indicated proteins in A549 cells infected with (+) or without (-) DENV and treated with the indicated concentration of OSU-T315 at 24 hpi are shown. BD, below detection.
Fig 5.
ILK regulates SOCS3 expression via Akt-Erk-NF-κB pathway.
(A and C) Representative western blots of phosphorylated (p-) and indicated proteins in mock- (M) and DENV-infected control (shLuc) and ILK knockdown (shILK) A549 cells at indicated hours post-infection (hpi) are shown. The ratios of phosphorylated to total p65 relative to mock-infected cells are shown. (B, D, and E) Representative western blots of indicated proteins in mock-infected (-) or DENV-infected (+) A549 cells treated with indicated concentrations of Bay 11–7082 (B), U0126 (D), or Akt inhibitor (E) at 24 hpi are shown. (F) The DENV yields in infected A549 cells treated with 10 μM of indicated inhibitors at 24 hpi are shown. Data represent mean + SD (error bars). **p < 0.01, ***p < 0.001.
Fig 6.
DENV NS1 and NS3 interact with ILK to attenuate IFN signaling.
(A) Representative western blots of indicated proteins in HA, HA-NS1, HA-NS3, and HA-NS5 expressing A549 cells are shown. (B and C) Representative western blots of indicated proteins (B) and quantitative RT-PCR of indicated genes (C) in HA, HA-NS1, and HA-NS3 expressing A549 cells at indicated hpi are shown. (D) A549 cells were transfected with Flag-ILK, HA, HA-NS1, HA-NS3, or HA-NS5 and co-immunoprecipitation performed by anti-HA or anti-Flag antibodies. Representative western blots of indicated proteins are shown. (E) Representative western blots of indicated proteins in control or infected cells after co-immunoprecipitation by anti-ILK antibody are shown. (F) A549 cells were transfected with Flag-ILK1-170, Flag-ILK171-452, HA-NS1 or HA-NS3 and co-immunoprecipitation performed by anti-Flag antibody. Representative western blots of indicated proteins are shown. (G) A549 cells were transfected with Flag-ILK, HA, HA-NS1 or HA-NS3 and co-immunoprecipitation performed by anti-Flag antibody. Representative western blots of indicated proteins are shown. The ratios of Akt co-immunoprecipitated with ILK to ILK precipitated by anti-Flag antibody are shown. Data represent mean + SD (error bars). ***p < 0.001. WCL, whole cell lysate.
Fig 7.
The inhibition of ILK reduces viral loads in the brains and mortality rate of DENV2-infected mice.
(A, B, C, and E) The 7-day-old C57BL/6 mice were mock-infected or infected with 6 × 105 plaque-forming unit (PFU) of DENV and treated without (control or -) or with 12.5 mg/kg OSU-T315. The disease scores (A), survival rate (B), viral loads in the mouse brains on day 7 post-infection (C), and levels of indicated proteins in the mouse sera day 7 post-infection (E) are shown. (D) The images of cortex regions stained with anti-NS3 (green) antibody from brain sections of mice mock- or DENV-infected and treated without (control) or with OSU-T315 on day 7 post-infection are shown. Nuclei were counterstained with DAPI. (F) The schematic diagram of mechanism mediated by ILK to promote DENV infection. This figure is created with Biorender.com. Data represent the mean ± SD (error bars) in panel A and the mean + SD (error bars) in panel E. Each symbol on the scattergram represents an individual sample. The black horizontal line represents the mean value for each group in panel C. *p < 0.01 and **p < 0.001 of 5 samples per data points via two-way ANOVA with the appropriate post-hoc test for multiple comparisons in panel A and E, via log-rank test in panel B, and via Student’s t test in panel C.