Moracin M inhibits EV71 infection primarily by blocking viral attachment to host cells

Figures

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  Fig 1. Screening of EV71 inhibitors using EV71-GFP. (A) RD cells were infected with EV71-GFP for 2 h in 12-well plates. Subsequently, the supernatant was replaced with fresh medium containing 31 candidate compounds, respectively (50 μM each). Ribavirin (80 μM) was used as a positive control, and 0.1% DMSO served as the vehicle control. The percentage of GFP-positive cells after 24 h of treatment was quantified by flow cytometry, and the relative inhibition rate of compound MM was calculated by comparing with the vehicle control, and the inhibition rate of SC-11 was compared with that of ribavirin. (B) Chemical structure of compound MM. RD cells (C) and Vero cells (D) pre-seeded in 96-well plates were treated with increasing concentrations of MM (0–200 μM) for 24 h. Cell viability was assessed using the CCK-8 assay, and the CC50 values of MM in RD cells (E) and Vero cells (F) were determined. All data are presented as mean ± SEM from three independent experiments. Statistical significance was evaluated using one-way ANOVA. P values are indicated as follows: *P < 0.05, ***P < 0.001, ****P < 0.0001; ns, not significant.

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  Fig 2. The effect of MM on EV71 infection. RD cells were infected with EV71-GFP (MOI = 1) for 2 h and subsequently treated with MM at concentrations of 0, 12.5, 25, 50 μM, or with 80 μM ribavirin. At 24 hpi, cytopathic effects and GFP-positive cells were observed under a microscope (A). The percentage of GFP-positive cells was quantified by flow cytometry, and the inhibition rate of EV71 infection (%) was calculated as [1 – FITC (treatment)/FITC (vehicle control)] × 100. IC50 values were determined based on the inhibition rates (B). Similar experiments were performed with Vero cells, and the IC50 of MM was measured and calculated (C). In parallel experiments, RD cells infected with EV71-GFP (MOI = 1) were treated with MM at concentrations of 0, 12.5, 25, and 50 μM. At 24 hpi, the supernatants were collected to determine TCID50 (D). EV71-GFP (MOI = 1) or EV71-LYG03 (MOI = 0.5) infected cells were treated with MM at concentrations of 0, 12.5, 25, and 50 μM. At 24 hpi, cells were harvested for the detection of EV71 RNA by RT-qPCR (E, G) and VP1 or 3D protein expression by Western blot (F, H), with GAPDH serving as an internal loading control. Magnification: 100× (scale bar: 100 μm). Both…

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  Fig 3. The influence of MM on the viral replication stage. (A) The diagram of the attachment, entry, and replication stages detection assay. (B) For the attachment assay, the pre-cooled RD cells were incubated with the mixture of EV71-GFP (MOI = 5) and 50 μM MM at 4°C for 1 h. After being washed with cold PBS, the viral RNA bound to the virus was determined by RT-qPCR. (C) For the entry assay, RD cells were incubated with EV71-GFP (MOI = 5) at 4°C for 1 h, were washed with PBS, and treated with 50 μM MM at 37°C for 1 h. The viral RNA was determined by RT-qPCR. (D) For the replication assay, RD cells were infected with EV71-GFP (MOI = 1) at 37°C for 2 h, and then the cells were washed and treated with 50 μM MM for 4 h. The cells were collected, and the viral RNA was detected by RT-qPCR. All data are presented as mean ± SEM of triplicate measurements. Statistical significance was evaluated using one-way ANOVA. P values are indicated as follows: **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

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  Fig 4. The effect of MM on virus or cells. RD cells were treated with 50 μM MM at 37°C for 1 h; after washing, they were infected with EV71-GFP (MOI = 1) for 24 h at 37°C. The proportion of GFP-positive cells and CPE was observed under a fluorescence microscope and bright field microscope, respectively (A), and viral RNA was quantified by RT-qPCR (B). EV71-GFP (MOI = 1) was incubated with 50 μM MM at 4°C for 4 h, was diluted 100-fold to minimize residual compound effects, and subsequently used to infect RD cells for 24 h. The proportion of GFP-positive cells and CPE was observed (C), and viral RNA (D) was detected. All data of viral RNA are presented as mean ± SEM of triplicate measurements. Magnification: 200× (scale bar: 50 μm). Statistical significance was evaluated using one-way ANOVA. P values are indicated as follows: ****P < 0.0001; ns, not significant.

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  Fig 5. The influence of MM on CA16 infection. (A) Vero cells were infected with CA16-#3 (MOI = 1) for 2 h, washed with PBS, and treated with different concentrations of MM. After 24 h of infection, the viral RNA was detected by RT-qPCR. (B) The expression of Vp1 protein was detected by Western blot. (C) The attachment phase was performed as in Fig. 3B. (D) The entry phase was performed as in Fig. 3C. (E) The replication phase was performed as in Fig. 3D. All data are presented as mean ± SEM of triplicate measurements. Statistical significance was evaluated using one-way ANOVA. P values are indicated as follows: **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

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  Fig 6. The evaluation of the acute toxicity of MM in newborn ICR mice. Three-day-old suckling mice were divided into three groups (6 mice per group): the PBS control group, the MM-treated group receiving 20 mg/kg or 60 mg/kg. The survival rate (A) and body weight changes (B) were monitored and recorded. Liver index (C), spleen index (D), and kidney index (E) were calculated as the ratio of individual organ weight to total body weight. Histopathological changes in the liver, spleen, and kidney were assessed by H&E staining (F). Magnification: 200× (scale bar: 50 μm).

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  Fig 7. The inhibitory effect of MM on EV71 infection in newborn ICR mice. Three-day-old mice were divided into three groups (n = 6). One group served as the negative control, which was treated with DMEM and PBS (DMEM + PBS). The other two groups were challenged with 10 μL of virus solution containing 10⁷ TCID₅₀ EV71-LYG03; subsequently, one group was administered MM at 20 mg/kg once daily for 5 consecutive days and designated as EV71 + MM group, while the other group received PBS and served as and designated as EV71 + PBS group. On day 6 post-infection, hindlimb muscles, lungs, and brain were collected from mice in all three groups. Viral RNA levels in hindlimb muscles, lungs, and brain (A) were quantified by RT-qPCR. Histopathological changes in hindlimb muscles, lungs, and brain were evaluated by H&E staining (B). The relative mRNA levels of IL-1β, IL-6, TNFα, and CXCL10 in these tissues were determined by RT-qPCR (C). Magnification: 200× (scale bar: 50 μm). All data are presented as mean ± SEM of triplicate measurements. Statistical significance was evaluated using one-way ANOVA and t-test. **P < 0.01, ***P < 0.001, ****P < 0.0001.