6-thioguanine inhibits EV71 replication by reducing BIRC3-mediated autophagy

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  Fig. 1. 6-TG inhibited EV71 infection. (A) The molecular structure of 6-TG was shown. (B) HT-29 cells were treated with serial concentrations (0-2000 µM) of 6-TG for 24 h, then the cell viability of the cells was determined by CCK8 assay. The viability of non-treated cells was considered as a blank control. The 6-TG-treated cell viability was determined as a percentage of the blank control. CC50 is 50% cytotoxicity concentration of 6-TG in HT-29 cells. (C) HT-29 cells were infected with EV71 at an MOI of 3 in the presence of 6-TG (0, 1, 3 µM). After 24 h, EV71 mRNA was quantified via quantitative real-time PCR, normalized against GAPDH. The EV71 VP1 protein expression was quantified via western blot analysis, normalized against β-actin. The intensity of western blot bands signals was quantified by ImageJ software (D). (E) HT-29 cells were infected with EV71 at an MOI of 3 for 4 h, then the cells were washed with PBS and incubated with 3 µM 6-TG. After 24 h, the culture supernatant was collected and added to Vero cells, the viral titers were calculated as the TCID50 after 72 h incubation. (F) HT-29 cells were infected with EV71 at an MOI of 3 in the presence of 6-TG at different…

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  Fig. 2. 6-TG inhibited EV71 replication at early stages of viral infection. (A) HeLa cells were treated with serial concentrations (0–50 µM) of 6-TG for 24 h, then the cell viability of the cells was determined by CCK8 assay. The viability of non-treated cells was considered as a blank control. The 6-TG-treated cell viability was determined as a percentage of the blank control. CC50 is 50% cytotoxicity concentration of 6-TG in HeLa cells. (B) HeLa cells were infected with EV71 at an MOI of 1 in the presence of 6-TG at different concentrations (0, 0.001, 0.01, 0.1, 1, 5 µM). After 24 h, the intracellular EV71 VP1 (green) was examined via ICW assay and normalized by DRAQ5 (red). The percent of EV71 inhibition was analyzed by Odyssey software, and the intracellular EV71 VP1 of EV71-infected cells without 6-TG treatment were considered as a negative control. (C) The design of action mode of time-of-addition assay. HeLa cells were infected with EV71 (MOI = 0.5) for 24 h, and 1 µM of 6-TG or DMSO were added to cells at -2, 0, 2, 4, 6, 8, 12 and 24 h of EV71 infection. After 24 h, the intracellular EV71 VP1 (green) was examined via ICW assay (D). The percent of inhibition of EV71 was analyzed…

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  Fig. 3. BIRC3 was the key regulator of 6-TG against EV71 infection. (A) HeLa cells were infected with EV71 at an MOI of 1 in the presence of NSC23766 at different concentrations (0, 5, 10, 30, 50, 100 µM). After 24 h, the intracellular EV71 VP1 (green) was examined via ICW assay and normalized by DRAQ5 (red). The percent of EV71 inhibition was analyzed by Odyssey software, and the intracellular EV71 VP1 of EV71-infected cells without NSC23766 treatment were considered as a negative control. (B) Identification of 14 common differential expressed genes (DEG) from untreated or 6-TG-treated HUVEC database (Control vs. 6-TG) and uninfected or EV71-infected mice database (GSE123550) (Mock vs. EV71) through SangerBox 3.0 online tools. Advanced Pie plot was performed using the OmicStudio tools at https://www.omicstudio.cn/tool. (C) The heatmap of the 14 common DEGs from Control vs. 6-TG group and Mock vs. EV71 group. Advanced Heatmap Plots was performed using the OmicStudio tools at https://www.omicstudio.cn. (D and E) HeLa cells were incubated with 1 or 3 µM 6-TG for 2 h. Then the BIRC3 mRNA was quantified by quantitative real-time PCR, normalized to GAPDH (D). The BIRC3 protein levels were…

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  Fig. 4. BIRC3 promoted EV71 replication and reversed the anti-EV71 effect of 6-TG. (A and B) HeLa cells were transfected with three specific siRNAs targeting BIRC3 (siBIRC3 #1, siBIRC3 #2, siBIRC3 #3) or control siRNA (siNC) for 30 h. The cells were harvested and the BIRC3 RNA levels were quantified via quantitative real-time PCR, normalized against GAPDH (A), the BIRC3 protein levels were evaluated by western blot assay, normalized to β-actin (B). (C and D) HeLa cells were transfected with siBIRC3 or siNC for 30 h, and then infected with EV71 at an MOI of 0.5 for 24 h. The cells were harvested and the EV71 RNA levels were quantified via quantitative real-time PCR, normalized against GAPDH (C). The EV71 VP1 protein expression was evaluated by western blot assay, normalized to β-actin. The intensity of western blot bands signals was quantified by ImageJ software (D). (E and F) HeLa cells were transfected with 1 µg plasmid encoding BIRC3 (pBIRC3) or control plasmid (pVector). After 30 h, the cells were collected, and the BIRC3 RNA levels were quantified via quantitative real-time PCR, normalized against GAPDH (E), the flag-BIRC3 protein expression was evaluated by western blot assay,…

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  Fig. 5. BIRC3 knockdown affected the cleavage function of EV71 2Apro and the expression of EV71 3AB. HeLa cells were transfected with plasmid GFP-2Apro, GFP-2B, GFP-3AB, GFP-3C, GFP-3D and HA-2C for 24 h, then were transfected with siBIRC3 or siNC for 30 h. The cells were harvested and the expression of eIF4G, GFP-tag, HA-tag was evaluated by western blot assay, normalized to β-actin or GAPDH. The intensity of western blot bands signals of cleaved eIF4G, GFP-2B/3AB/3 C/3D, HA-2 C, β-actin and GAPDH were quantified by ImageJ software. Data were pooled from three independent experiments, and were shown as the mean ± SEM (ns, no significant, *P < 0.05, **P < 0.01)

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  Fig. 6. 6-TG suppressed EV71 replication by diminishing BIRC3-mediated autophagy. (A) HeLa cells were Mock-infected or EV71 (MOI = 0.5) infected for 12 h, then cells were fixed for immunostaining using EV71 2C antibody (red). Nuclei were labeled with DAPI (blue). Scale bars, 10 μm. The white arrows showed the formed GFP-LC3 puncta. (B) HeLa cells were infected with EV71 at an MOI of 0.5, and the cells were harvested at 12, 24 hpi (hours post infection). The EV71 VP1, LC3 I, LC3 II and P62 expression was quantified via western blot analysis, normalized against β-actin. The intensity of western blot bands signals of LC3 II/LC3 I and P62/β-actin was quantified by ImageJ software. (C) HeLa cells were infected with EV71 at an MOI of 0.5 in the presence of 1 µM 6-TG. After 24 h, the EV71 VP1, LC3 I, LC3 II and P62 expression was quantified via western blot analysis, normalized against β-actin. The intensity of western blot bands signals of LC3 II/LC3 I and P62/β-actin was quantified by ImageJ software. (D) HeLa cells were transfected with siBIRC3 or siNC for 30 h, then the cells were infected with EV71 at an MOI of 0.5 for 24 h. The BIRC3, LC3 I, LC3 II, P62 and EV71 VP1 expression in…

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  Fig. 7. 6-TG addition diminished BIRC3-mediated autophagy. (A) HeLa cells were incubated with 1 or 3 µM 6-TG for 24 h, the LC3 I, LC3 II, P62 and BIRC3 expression was quantified via western blot analysis, normalized against β-actin. The intensity of western blot bands signals of LC3 II/LC3 I and P62/β-actin was quantified by ImageJ software. (B) HeLa cells were stimulated with 100 nM rapamycin for 12 h, then they were incubated with 1 µM 6-TG for 24 h. The LC3 I, LC3 II and P62 expression was quantified via western blot analysis, normalized against β-actin. The intensity of western blot bands signals of LC3 II/LC3 I and P62/β-actin was quantified by ImageJ software. (C) HeLa cells were transfected with 0.5–1 µg pBIRC3 or pVector for 30 h. The cells were harvested and Fag-BIRC3, LC3 I, LC3 II and P62 expression was evaluated by western blot assay, normalized to β-actin. The intensity of western blot bands signals of LC3 II/LC3 I and P62/β-actin was quantified by ImageJ software. (D) HeLa cells were transfected with siBIRC3 or siNC for 30 h, respectively. The cells were harvested and BIRC3, LC3 I, LC3 II and P62 expression was evaluated by western blot assay, normalized to β-actin. The…