The Pyrimidine Analog FNC Potently Inhibits the Replication of Multiple Enteroviruses

Figures

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  FIG 1. Chemical structures of the eight nucleoside analogs used in this study.

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  FIG 2. Cytotoxicity and anti-EV71 activity of FNC and its 7 analogs. (A) The dose-dependent cytotoxicity profile of the 8 compounds was detected using a CCK-8 kit in RD cells at 48 h. (B) Anti-EV71 activities of the 8 compounds in the safe-dose range. RD cells were infected with EV71 at an MOI of 0.1 for 4 h, washed twice with DMEM, and supplemented with DMEM containing the indicated concentration of the 8 compounds for another 48 h. The cells were harvested for Western blot (WB) analysis, and tubulin was used as a loading control. (C) Cytotoxicity of FNC in RD cells was detected at 0 to 1 μM at 48 h. (D) The CC50 of FNC in RD cells was calculated using GraphPad Prism7.

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  FIG 3. FNC potently inhibits the replication of EV71 in a dose-dependent manner. (A and B) Inhibitory effect of FNC on EV71 replication in the 0.01 to 1,000 nM (A) or 10 to 100 nM (B) dose range. RD cells were infected with EV71 as described in Fig. 2B, and the indicated concentration of FNC was added to RD cells for 48 h. The cells were then harvested for WB analysis. The supernatant from the infected RD was centrifuged and then loaded for WB analysis. Tubulin was used as a loading control. (C) Cellular EV71 RNA levels in panel B were detected by RT-qPCR. GAPDH was used as a control. The EV71 RNA level without FNC treatment was set as 100%. (D) The EC50 of FNC was calculated according to panel C using GraphPad Prism7. (E and F) Viral titers in the supernatants from the experiment shown in panel B were determined by the plaque assay (E) and by the cytopathic effect (CPE) method (F). The results shown are the means with SDs from two independent experiments. The asterisks indicate statistically significant differences between groups, as assessed by Student's t test (*, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001; ns, not significant).

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  FIG 4. FNC potently inhibits the replication of CA16 and CA6 in a dose-dependent manner. (A and G) A 100 nM dose of FNC inhibited CA16 (A) and CA6 (G) viral replication in RD cells. (B) The inhibitory effect of FNC on CA16 replication in the 10 to 200 nM dose range. The CA16 VP1 protein in cell lysate or culture supernatant was detected by WB analysis, and tubulin was used as a loading control. (C) CA16 RNA levels in the cells from the experiments shown in panel B were detected by RT-qPCR. GAPDH was used as a control. The CA16 RNA level without FNC treatment was set as 100%. (D) The EC50 of FNC was calculated according to panel C using GraphPad Prism7. (E and F) The viral titers in the supernatants from the experiment shown in panel B were determined by the plaque assay (E) and by the cytopathic effect (CPE) method (F). (H) The inhibitory effect of FNC on CA6 replication in the 10 to 100 nM dose range. The CA6 VP1 protein in cell lysate or culture supernatant was detected by WB analysis, and tubulin was used as a loading control. (I) CA6 RNA levels in the cells from the experiments shown in panel H were detected by RT-qPCR. GAPDH was used as a control. The CA6 RNA level without FNC…

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  FIG 5. FNC potently inhibits the replication of CVB3 and EVD68 in a dose-dependent manner. (A and G) A 100 nM dose of FNC inhibited CVB3 (A) and EVD68 (G) viral replication in RD cells. (B) The inhibitory effect of FNC on CVB3 replication is in the 10 to 100 nM dose range. The CVB3 VP1 protein in cell lysate or the culture supernatant was detected by WB analysis, and tubulin was used as a loading control. (C) CVB3 RNA levels in the cells from the experiments shown in panel B were detected by RT-qPCR. GAPDH was used as a control. The CVB3 RNA level without FNC treatment was set as 100%. (D) The EC50 of FNC was calculated according to panel C using GraphPad Prism7. (E and F) Viral titers in the supernatants from the experiment shown in panel B were determined by the plaque assay (E) and by the cytopathic effect (CPE) method (F). (H) The inhibitory effect of FNC on EVD68 replication in the 10 to 100 nM dose range. The EVD68 VP1 protein in cell lysate or culture supernatant was detected by WB analysis, and tubulin was used as a loading control. (I) EVD68 RNA levels in the cells from the experiments shown in panel H were detected by RT-qPCR. GAPDH was used as a control. The EVD68 RNA level…

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  FIG 6. FNC has no inhibitory effect on RSV and IAV. (A and B) Cytotoxicity of FNC in HEK293T (A) or MDCK (B) cells was detected using a CCK-8 kit at 48 h. (C) HEK293T cells were infected with RSV at an MOI of 0.5 for 4 h, washed twice with DMEM, and supplemented with DMEM containing the indicated concentration of FNC for another 48 h. The cells were harvested. Representative structural proteins G, N, M, and M2-1 were detected by WB analysis, and tubulin was used as a loading control. (D) RSV RNA levels in cells were detected by RT-qPCR. GAPDH was used as a control. The RSV RNA level without FNC treatment was set as 100%. (E) MDCK cells were infected with IAV H1N1 at an MOI of 0.1 for 4 h, washed twice with DMEM, and supplemented with DMEM containing the indicated concentration of FNC for another 48 h. The cells were harvested for WB analysis, and tubulin was used as a loading control. (F) IAV RNA levels in cells were detected by RT-qPCR. GAPDH was used as a control. The IAV RNA level without FNC treatment was set as 100%.

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  FIG 7. FNC inhibits the transcription of EVs through interacting with 3Dpol. (A to E) FNC inhibited both positive- and negative-strand RNA production of EV71 (A), CA16 (B), CA6 (C), CVB3 (D), and EVD68 (E). RD cells were infected with the indicated EVs, treated with the indicated concentration of FNC for 48 h, and harvested for RNA extraction and RT-qPCR. GAPDH was used to normalize the cells. The asterisks indicate statistically significant differences between groups, as assessed by Student's t test (*, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001; ns, not significant).

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  FIG 8. FNC inhibits the activity of 3Dpol by direct interaction. (A) ITC binding curves for EV71 3Dpol protein and negative control (buffer) or FNC or ribavirin at 25°C showed that FNC binds to EV71 3Dpol with a Kd of 6 × 10−6 mol/liter. The titrant of 1.0 mM FNC or 3 mM ribavirin was mixed with 100 μM EV71 3Dpol protein solution. Curves in the bottom graphs show the fitting of data to a one-set model by the nonlinear Levenberg-Marquardt fitting algorithm. (B) Fluorescence-based polymerization assay using EV71 3Dpol. Experiments were carried out as described in Materials and Methods. The results shown are the means with SDs from three independent experiments. A.U., arbitrary units.

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  FIG 9. FNC protects neonatal mice from EV71 and CA16 lethal challenges. (A to F) One-day-old ICR mice (n = 8 to 10) were inoculated with 10 μl of EV71 CC063 at 105.5 CCID50 ml−1 (A, C, and E) or CA16 CC045 at 102.7 CCID50 ml−1 (B, D, and F) and at 1 h postchallenge injected intraperitoneally with FNC at days 1, 3, 6, 9, and 12. Clinical scores and survival rates were monitored for 15 days postchallenge. Various grades of clinical disease were identified as follows: 0, healthy; 1, lethargy and inactivity; 2, wasting; 3, limb-tremor weakness; 4, hind-limb paralysis; and 5, moribund or dead. (E and F) Viral loads of EV71 (E) and CA16 (F) in samples of the brain, lung, spine skeletal muscle, and hind-limb muscle tissues of infected mice were assessed by RT-qPCR at day 8. The results represent the mean virus loads (log10 copies/mg tissue) ± SDs (three mice per group, repeated three times).

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  FIG 10. Pathology analysis of EV71- or CA16-infected neonatal mice. One-day-old ICR mice were intracerebrally inoculated with EV71 CC063 (105.5 CCID50/ml), CA16 CC045 (102.7 CCID50/ml), or DMEM (mock control). At 1 h postchallenge, FNC or DMSO was injected intraperitoneally, as described in Materials and Methods. (A and B) Representative images from the lung (a to c), brain (d to f), spine skeletal muscle (g to i), and hind-limb muscle (j to l) postinfection are shown for EV71-infected (A) or CA16-infected (B) mice. The images for noninfected mice were used as a control (a, d, g, and j in panels A and B). Magnification, ×400.