Fig 1.
Inhibition of SKI-1/S1P enzymatic activity using PF-429242 impairs activation of the SREBP pathway and correlates with a dramatic decrease in lipid droplet number and area.
(A–D) Huh-7.5.1 cells were treated with various concentrations of PF-429242 and corresponding concentrations of DMSO for 24 hours before the inhibitor was removed and fresh complete media was added to the cells for an additional 48 hours. (A) The relative cytotoxicity of the compound was determined using an MTS-based cell viability assay (CC50 = 81 μM). The absorbance measured at 490 nm is proportional to the number of living cultured cells. (B) Total RNA was extracted, and the mRNA levels of SREBP-1c, SREBP-2, PCSK9, LDLR, FURIN, and SKI-1/S1P were quantified by qRT-PCR. Results were normalized against β-actin mRNA levels and expressed as fold change. Statistical significance was calculated for PF-429242-treated cells (20 μM) compared to 0.02% DMSO-treated cells (control) with a two-way ANOVA for each mRNA presented. (C) Representative images of the effect of PF-429242 (20 μM) and 0.02% DMSO on lipid droplets are shown. Fixed cells were permeabilized with Triton X-100 and stained for cell nuclei using Hoechst dye and for lipid droplets using Nile red. Images were obtained using a Leica SP8 confocal microscope with a 100x objective. (D) Abundance of LDs was quantified by measuring the average LD-positive area (μm2)/cell and the average number of LDs/cell based on Nile red fluorescence in untreated, 0.02% DMSO-treated, and PF-429242-treated cells (20 μM) using Fiji software (>50 cells analyzed). Statistical significance was calculated with a two-way ANOVA with a Bonferroni’s post-test. Values represent average ± SEM of three independent experiments (****, p < 0.001).
Fig 2.
Huh-7.5.1 cells support DENV-2 replication and DENV capsid protein binding to hepatic lipid droplets.
(A–E) Huh-7.5.1 cells were infected with DENV-2 (MOI = 1) or mock-infected. (A) Total RNA was extracted at various time-points (0, 4, 8, 24, 48, and 72 h) post-infection, and mRNA expression of DENV-2 RNA was quantified by qRT-PCR. Results were normalized against control β-actin mRNA levels and expressed as fold change relative to the time-matched mock-infected controls. (B–E) Cells were harvested at 24h and 48h post-infection. Cells were fixed with paraformaldehyde and permeabilized with Triton X-100 (B) or digitonin (C). (D) DENV C accumulation around lipid droplets in Huh-7.5.1 cells infected with DENV (MOI = 1) at 48 hpi. Fixed cells were stained for cell nuclei using Hoechst dye (blue), for lipid droplets using Nile red (red), and for DENV using anti-capsid (green). (E) Abundance of LDs was quantified by measuring the average LD-positive area (μm2)/cell and the average number of LDs/cell based on Nile red fluorescence in mock-infected cells, DENV-2 infected cells (24 hpi), and DENV-2 infected cells (48 hpi) using Fiji software (>50 cells analyzed). Images were obtained using a Leica SP8 confocal microscope with a 100x objective. Results (mean ± SEM) from at least three independent experiments are shown. Statistical significance was calculated for DENV-2-infected cells compared to mock-infected cells at time-points corresponding to DENV-2-infected cells with a two-way ANOVA with a Bonferroni’s post-test (*, p < 0.05; ****, p < 0.001).
Fig 3.
Pretreatment of Huh-7.5.1 cells with PF-429242 results in a dose-dependent decrease in intracellular DENV-2 NS1 protein abundance and a 3-log decrease in extracellular viral titer.
(A–B) Huh-7.5.1 cells were either untreated or treated with increasing concentrations of PF-429242 for 24 hours. The inhibitor was removed and the cells were then infected with DENV-2 (MOI = 0.01) for 48 hours. (A) Cell lysates were probed for DENV NS1 (green) and normalized to β-tubulin (red). Representative Western blot for the effect of PF-429242 on DENV NS1 protein level is shown. (B) Dose response curve of normalized, averaged NS1 signal quantified from Western blots of three independent experiments (EC50 = 1.2 μM). (C) Huh-7.5.1 cells were treated with increasing concentrations of PF-429242 or with 0.03% DMSO (control) for 24 hours. The inhibitor was removed and the cells were then infected with DENV-2 (MOI = 0.01). 48 hours post-infection, supernatant was collected and viral titer was determined by infecting naïve Vero E6 cells and counting plaques 5 days post-infection. Results (mean ± SEM) from three independent experiments are shown. Statistical significance was calculated for PF-429242-treated cells compared to control with a ratio paired Student’s t-test (C) (**, p < 0.01).
Fig 4.
Pretreatment of Huh-7.5.1 cells with PF-429242 results in a robust decrease in intracellular DENV-2 RNA and post-treatment of DENV-2 infected Huh-7.5.1 cells with PF-429242 impairs the assembly and/or release of infectious virus particles.
(A–B) Huh-7.5.1 cells were treated either with 20 μM AcPF-429242, with various concentrations of PF-429242 (10–30 μM), or DMSO (0.01–0.03%) (control) for 24 hours. The inhibitor was removed and the cells were then infected with DENV-2 (MOI = 0.01) for 48 hours. (A) Total RNA was harvested and DENV-2 RNA levels, normalized to β-actin transcript levels, were relatively quantified in cell extracts using qRT-PCR. (B) Collected supernatant was cultured with naïve Huh-7.5.1 cells for 48 hours, and DENV-2 RNA levels were quantified. (C–E) Huh-7.5.1 cells were infected with DENV-2 (MOI = 0.01). 24 hours post-infection, cells were treated either with 0.03% DMSO (control), 20 μM AcPF-429242, or 20/30 μM PF-429242 for 48 hours. Total intracellular RNA during primary infection (C) and secondary infection (D), and extracellular RNA during primary infection (E), were harvested and analyzed for DENV RNA levels. Intracellular DENV-2 RNA levels (C, D) were normalized to β-actin transcript levels, while extracellular DENV-2 RNA levels (E) were normalized by volume and then relatively quantified using qRT-PCR. Values represent mean ± SEM of three independent experiments. Statistical significance was calculated compared to control with a one-way ANOVA with a Bonferroni’s post-test (**, p < 0.01; ***, p < 0.005).
Fig 5.
Extracellularly applied oleic acid, an inducer of lipid droplet formation, rescues intracellular DENV-2 RNA abundance in PF-429242-treated Huh-7.5.1 cells.
(A–D) Huh-7.5.1 cells were treated either with 0.01% DMSO (control) or 10 μM PF-429242 for 24 hours. The inhibitor was removed and the cells were then infected with DENV-2 (MOI = 0.01) (A–B) or mock-infected (C–D) with or without addition of 0.6 mM oleic acid (with BSA in molar ratio 6:1) for 24 hours. (A) Total RNA was harvested and DENV-2 RNA levels, normalized to β-actin transcript levels, were relatively quantified in cell extracts using qRT-PCR. (B) Collected supernatant was cultured with naïve Huh-7.5.1 cells for 48 hours, and DENV-2 RNA levels were quantified. (C) Representative images of the effect of PF-429242 and oleic acid on lipid droplets are shown. Fixed cells were permeabilized with Triton X-100 and stained for cell nuclei using Hoechst dye and for lipid droplets using Nile red. Images were obtained using a Leica SP8 confocal microscope with a 100x objective. (D) Abundance of LDs was quantified by measuring the average LD-positive area (μm2)/cell and the average number of LDs/cell based on Nile red fluorescence in 0.01% DMSO-treated (control), PF-429242-treated (10 μM), OA/BSA-treated, and PF-429242 (10 μM) + OA/BSA-treated cells using Fiji software (>50 cells analyzed). Statistical significance was calculated with a two-way (A, D) and one-way (B) ANOVA with a Bonferroni’s post-test (*, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001). Values represent mean ± SEM of three independent experiments.
Fig 6.
Pretreatment of Huh-7.5.1 cells with PF-429242 results in a robust decrease in intracellular viral RNA for all four DENV serotypes.
Huh-7.5.1 cells were treated either with 0.02% DMSO (control), 20 μM AcPF-429242, or 20 μM PF-429242 for 24 hours. The inhibitor was removed and the cells were then infected with one of the four DENV serotypes (MOI = 0.01) for 48 hours. Total RNA was harvested and DENV RNA levels of four serotypes, normalized to β-actin transcript levels, were relatively quantified in cell extracts using qRT-PCR. Values represent mean ± SEM of three independent experiments. Statistical analysis was performed for PF-429242 and AcPF-429242 treated cells compared to control with a two-way ANOVA with a Bonferroni’s post-test (**, p < 0.01; ***, p < 0.005).