Voxtalisib inhibits enterovirus 71 replication by downregulating host RAN and restoring IFN–STAT signaling

Fig. 1. Voxtalisib inhibits EV71 replication in vitro. (A) Chemical structure of voxtalisib. (B, C) Cytotoxicity of voxtalisib in RD and HeLa cells assessed via the MTT assay. (B). Ribavirin CC50 values in RD and HeLa cells. (C). (D) Western blot and (E) immunofluorescence(scale bar: 100 μm) analysis of VP1 expression in RD cells infected with EV71 (MOI 0.1, 2 h) and treated with voxtalisib or ribavirin. (F, G) Culture supernatants were collected, subjected to freeze–thaw cycles, diluted, and analyzed using plaque (F) and TCID50 assays (G). (H, I) VP1 protein and EV71 mRNA levels 8, 12, 18, and 20 h post-treatment. Abbreviations: EV71, enterovirus 71; RD, rhabdomyosarcoma; CC50, cytotoxic concentration 50; MOI, multiplicity of infection; VP1, viral protein; TCID50, tissue culture infective dose 50.

Fig. 2. Voxtalisib inhibits the post-entry stage of EV71 replication. (A) Virucidal assay: Voxtalisib and EV71 were preincubated at 4 ℃ for 1 h or added separately before RD cell infection (1 h, 37 ℃). (B) EV71 vRNA levels 20 h post-infection (two-way ANOVA). (C) Binding/entry assay. (D) RD cells were treated with varying voxtalisib concentrations and incubated with EV71 (MOI = 0.1, 4 ℃, 1 h). Bound EV71 was analyzed using RT-qPCR. (E) Cells were incubated with EV71 (MOI = 0.1, 4 ℃, 1 h), washed, and treated with voxtalisib (37 ℃, 1 h), followed by quantification of EV71 vRNA levels. (F) RD cells were infected with EV71 (2 h), treated with voxtalisib (20 h), and analyzed via RT-qPCR. (H) IC50 values were determined based on vRNA inhibition rates, and SI was calculated as CC50/IC50. Data are presented as mean ± standard deviation (SD). Group comparisons were performed using one-way ANOVA. All experiments were conducted in triplicate (n = 3). Significance was defined as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Abbreviations: CC50, cytotoxicity concentration 50; EV71, enterovirus 71; RD, rhabdomyosarcoma; MOI, multiplicity of infection; RT-qPCR, quantitative…

Fig. 3. Voxtalisib treatment reduces RAN levels, and changes in RAN expression levels affect VP1 expression. (A) Venn diagrams were constructed for Group I vs. C, Group T vs. I, Group D vs. C, and the GeneCards database to identify key proteins associated with EV71 infection and voxtalisib treatment. (B) RAN mRNA and protein levels in RD cells treated with voxtalisib (4 μmol/L) for 8, 12, 18, and 24 h. (C) RAN mRNA and protein expression in RD cells treated with increasing voxtalisib concentrations (37 ℃, 24 h). (D) RAN mRNA and protein levels in RD cells infected with varying EV71 MOIs for 22 h. (E) RAN mRNA and protein levels in RD cells infected with EV71 (37 ℃, 2 h) and treated with increasing voxtalisib concentrations (20 h). (F) VP1 mRNA and protein levels in RD cells with RAN knocked down via siRNA and infected with EV71. (G) VP1 mRNA and protein levels in RD cells overexpressing RAN and infected with EV71 (MOI 0.1). (H) Western blot analysis of VP1 and RAN protein levels in RD cells transfected with si-NC or si-RAN, infected with EV71 (MOI = 0.5), and treated with or without voxtalisib (4 μmol/L). RAN knockdown reduced VP1 expression, and voxtalisib did not further suppress VP1…

Fig. 4. RAN regulates IFN–STAT signaling and inhibits EV71 replication. (A) Nuclear and cytoplasmic RAN, p-STAT1/2, and STAT1/2 proportions in RD cells infected with EV71 (2 h) and treated with voxtalisib (37 ℃, 22 h). (B) RAN, p-STAT1/2, and STAT1/2 levels in different RD cell fractions following treatment with or without voxtalisib (37 ℃, 22 h) and IFN-α (10 ng/mL). (C) Expression of ISGs in RD cells treated with or without voxtalisib (37 ℃, 22 h) and IFN-α (10 ng/mL). (D) Nuclear and cytoplasmic RAN, p-STAT1/2, and STAT1/2RD in RD cells treated with or without voxtalisib (37 ℃, 22 h), Leptomycin B (10 ng/mL), and IFN-α (10 ng/mL). (E) RAN, p-IRF3/7 and NF-κB levels in different RD cell fractions treatment with or without voxtalisib (37 ℃, 22 h) and IFN-α (10 ng/mL). (F) VP1 and RAN protein expression in RD cells transfected with a RAN overexpression plasmid or empty vector and infected with EV71 (MOI = 0.1, 22 h). (G) Plaque assay of viral titers in culture supernatants. (H) RD cells were transfected with FLAG–RAN, followed by treatment with IFN-α (10 ng/mL for 30 min). Co-immunoprecipitation was performed using anti-FLAG or control IgG antibodies, and precipitated proteins were…

Fig. 5. Voxtalisib inhibits EV71 replication in vivo. (A) Schematic representation of the animal experiment workflow. Two-day-old ICR suckling mice infected with EV71 were treated with vehicle or voxtalisib (0.25 mg/kg) daily for 8 days. (B) Survival curves following EV71 infection and voxtalisib treatment. (C) Body weight growth curves of mice infected with EV71 and treated with voxtalisib. (D–H) Viral loads in the heart, liver, spleen, brain, and forelimb tissues of mice. (I) Representative H&E staining of the liver and spleen tissues following EV71 infection and voxtalisib treatment. Data are presented as mean ± standard deviation (SD). Group comparisons were performed using one-way ANOVA. All experiments were conducted in triplicate (n = 3). Significance was defined as follows: *p < 0.05, **p < 0.01 (scale bar: 100 μm). Abbreviations: EV71, enterovirus 71; H&E, hematoxylin and eosin.

Fig. 6. Histopathological assessment of major organs in suckling mice treated with voxtalisib. H&E staining of tissue sections from heart, liver, spleen, brain, and front leg skeletal muscle collected from ICR suckling mice following daily intraperitoneal administration of voxtalisib (0.25 mg/kg) for 0–6 days. No evidence of necrosis, edema, inflammatory infiltration, or structural disruption was observed in the heart, spleen, brain, or muscle tissues throughout the treatment period. Mild hepatic steatosis was noted in some liver sections, consistent with common metabolic responses to small-molecule compounds. All images were captured at 200× magnification (scale bar: 50 μm).

Fig. 7. Voxtalisib inhibits the replication of various enteroviruses. (A) Cells infected with CVB3 (A, E), CVB4-5 (B, F), CVB4-7 (C, G), and Echo11 (D, H) (MOI 0.1, 2 h) and treated with voxtalisib for 20 h. Viral vRNA expression was measured (A–D), and virus titers were determined (E–H). Data are presented as mean ± standard deviation (SD). Group comparisons were performed using one-way ANOVA. All experiments were conducted in triplicate (n = 3). Significance was defined as follows: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (one-way ANOVA). Abbreviations: CVB, Coxsackie B viruses; Echo11, Echovirus 11; MOI, multiplicity of infection.