Gemcitabine, a broad-spectrum antiviral drug, suppresses enterovirus infections through innate immunity induced by the inhibition of pyrimidine biosynthesis and nucleotide depletion

Figures

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  Figure 1. The effect of exogenous nucleosides on the anti-CVB3 activity of gemcitabine (A) HeLa cells were infected with CVB3 and simultaneously treated with various concentrations of gemcitabine and 100 μM of 4 nucleosides. At 8 hours post-infection the virus-infected cells were visualized by staining with anti-CVB3 3Cpro antibody, and the percentage of infected cells among total cells was calculated by setting the value from DMSO-treated cells as 100%. The average and standard deviation were obtained from three independent experiments. *** indicates P < 0.001. (B) HeLa cells treated with the indicated concentrations of gemcitabine and 4 nucleosides without CVB3 infection were also analyzed for the cell viability by using the MTT assay.

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  Figure 2. The effect of exogenous nucleobases and intermediates of pyrimidine biosynthetic pathway on the anti-CVB3 activity of gemcitabine Four nucleobases (adenine, guanine, cytosine and uracil) and intermediates (dihydroorotate, orotic acid and UMP) (100 μM) of pyrimidine biosynthetic pathway were treated as described in Figure 1A (A) and in Figure 1B (B). Cytidine was used as a positive control. The average and standard deviation were obtained from three independent experiments. *** indicates P < 0.001. (C) Pyrimidine biosynthetic pathway. Tested metabolites were indicated as boxes and metabolites with the significant effect were shown as orange boxes. The salvage pathway affected by gemcitabine was depicted by blue arrows.

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  Figure 3. The effect of gemcitabine on ISRE promoter HeLa cells were transfected with plasmid encoding ISRE-luciferase and then treated with various concentrations of gemcitabine or IFN-α. At 8 or 24 hours after compound treatment cells were assayed for the firefly luciferase activity. (A) Gemcitabine (8h), (B) IFN-α (8h), (C) Gemcitabine (24h), and (D) IFN-α (24h). The relative luciferase activities in percentage were calculated by setting the value from DMSO-treated cells as 100%. The average and standard deviation were obtained from three independent experiments.

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  Figure 4. The effect of gemcitabine on ISGs HeLa cells were treated with various concentrations of gemcitabine (A) or IFN-α (B). After 24 hours, cellular RNAs were isolated and the expression levels of 5 ISGs (CXCL10, IRF7, IRF9, IFIT1 and DDX58) were quantified using real-time PCR. Results were normalized as a percentage relative to DMSO-treated cells. The average and standard deviation were obtained from three independent experiments.

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  Figure 5. Suppression of gemcitabine-induced ISG expression by exogenous cytidine HeLa cells were treated with gemcitabine (20 μM) or IFN-α (10 ng/ml) in the presence or absence of cytidine (50 μM). After 24 hours, cellular RNAs were isolated and the expression of IFIT1 (A) and DDX58 (B) mRNAs was determined by quantitative real-time PCR. Results were normalized as a percentage relative to DMSO-treated cells. The average and standard deviation were obtained from three independent experiments. * indicates P < 0.05.