Identification of a novel binding inhibitor that blocks the interaction between hSCARB2 and VP1 of enterovirus 71

Figures

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  Fig. 1. Structure of EV-A71 inhibitors, PTC-209HBr and derivatives.

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  Fig. 2. Antiviral effect of PTC-209HBr and enviroxime in vitro. (A–H) PTC-209HBr or enviroxime significantly inhibited viral RNA level accumulation; viral protein expression and robustly impeded viral-caused CPE. Vero or RD cells were pretreated with EV-A71 at an MOI of 0.1 and then cells were incubated at 4 °C for 1 h, followed by treatment of the indicated concentration of PTC-209HBr or enviroxime for 24 h at 1 hpi. Relative viral RNA levels and protein levels in dose-dependent manner were analyzed by qRT-PCR (A, B) and Western blot (C, D), respectively. Further antiviral efficacy of PTC-209HBr against EV-A71 was tested by IF in Vero cells (E) and a CPE assay in RD cells (F). Further antiviral effects of enviroxime against EV-A71 were tested by IF in Vero cells (G) and CPE assay in RD cells (H).

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  Fig. 3. Antiviral effect of PTC-209HBr and enviroxime against the selected specieses of Enterovirus (genus). (A, B) Concentration-response profiles used for EC50 determination are shown for enterovirus inhibitors (A) PTC-209HBr and (B) enviroxime. Data points are means of triplicates SEM, for curve-fitting, described in Materials and Methods. One representative dataset of at least two independent experiments is shown. (C) EC50s of PTC-209HBr and enviroxime for selected enterovirus species were determined in short-term treatment assay by CPE (Materials and Methods). Representative EC50 values from 2 to 3 independent experiments are given.

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  Fig. 4. Selection of PTC-209HBr resistance and sensitivity to VP1 mutants. (A) The PTC-209HBr resistance profiling. (B) The sensitivity of PTC-209HBr to VP1 mutant viruses, which was determinated by a CPE assay. (C) EC50s of PTC-209HBr for VP1 mutant EV-A71 were determined in short-term treatment assay by CPE. (D) The sensitivity of PTC-209HBr to VP1 to PTC-209HBr at a concentration of 3.56 μM was determined by a plaque assay. For the plaque assay, RD cells were infected with EV-A71 or mutants at an MOI of 2 × 10−4. For the CPE assay, RD cells were infected with EV-A71 at an MOI of 0.1.

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  Fig. 5. Binding affinities of PTC-209HBr, derivatives, pirodavir and enviroxime among VP1 or VP1 mutants were determined by MST. (A) The change in fluorescence to thermophoresis at the increasing concentrations of PTC-209HBr (4.8–156000 nM) in the presence of 500 nM VP1-EGFP. (B) The binding affinities of PTC-209HBr, derivatives (12s, 12t), pirodavir and enviroxime among VP1 or VP1 mutants.

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  Fig. 6. The action stage of PTC-209HBr, enviroxime and pirodavir was determined by a time-course assay. PTC-209HBr, enviroxime and pirodavir at 1.78 μM, 0.56 μM and 0.72 μM were added to the EV-A71-infected RD or Vero cells at the indicated time points. Samples were harvested at 12 hpi as described in the Materials and Methods. (A) Western blotting was carried out to assess the accumulation of VP1 expression in the presence of PTC-209HBr, enviroxime and pirodavir (A, B). The relative EV-A71 RNA levels in cells of the treated or untreated cases with PTC-209HBr, enviroxime and pirodavir were determined by qRT-PCR. One set of representative data out of at least two independent experiments with standard errors are showed.

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  Fig. 7. PTC-209HBr impedes EV-A71 binding and entry. The binding and entry assays were quantitated by qRT-PCR or Western blot. PTC-209HBr, enviroxime and pirodavir at specific concentration were added to the EV-A71-infected RD or Vero cells at the indicated time points. Samples were harvested at indicated time points as described in the Materials and Methods. (A) qRT-PCR was carried out to assess relative EV-A71 RNA levels in cells of the treated or untreated cases with PTC-209HBr (3.2 μM), enviroxime (0.32 μM) and pirodavir (0.64 μM) during the binding stage. (B) PTC-209HBr decreased EV-A71 infection in a dose-independent manner during the binding stage. (C) qRT-PCR was carried out to assess relative EV-A71 RNA levels in cells of the treated or untreated cases with PTC-209HBr (3.2 μM), enviroxime (0.32 μM) and pirodavir (0.64 μM) during the entry stage. (D) PTC-209HBr dose-dependently decreased EV-A71 infection during the entry stage. (E) The accumulation of VP1 expression in the presence of PTC-209HBr, enviroxime and pirodavir was analyzed by a Western blot assay during the binding stage. One set of representative data out of at least two independent experiments with standard errors…

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  Fig. 8. Colocalization of EV-A71 VP1 and hSCARB2 in the presence of PTC-209HBr, enviroxime and pirodavir. (A) The colocalization of EV-A71 VP1 and hSCARB2 in the presence of PTC-209HBr, enviroxime or pirodavir. The yellow square indicates the colocalization of EV-A71 VP1 and hSCARB2. EV-A71 was mixed with the indicated concentration of PTC-209HBr and then incubated at 37 °C for 1 h. Then, the mixtures were incubated with RD cells at 4 °C for 1 h, washed with cold PBS three times, fixed with 10% formaldehyde solution and further subjected to IF analysis. These data were acquired from at least two independent assays. (B) Inhibition of the interaction between VP1 and hSCARB2 by PTC-209HBr was analyzed by Co-IP. HEK293T cells were co-transfected with Flag-VP1 and HA-hSCARB2, followed by the treatment of PTC-209HBr or DMSO at 24h post transfection. Cell lysates were immunoprecipitated with an anti-Flag antibody and were then analyzed by a Western blot assay.

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  Fig. 9. Colocalization of EV-A71 variants and hSCARB2 in the treatment of PTC-209HBr or DMSO. The yellow square indicates the colocalization of VP1 and hSCARB2. EV-A71 was mixed with the indicated concentration of PTC-209HBr and then incubated at 37 °C for 1 h, followed by incubation with RD cells at 37 °C for 1 h. The treated cells were washed with cold PBS five times and then incubated at 37 °C and 5% CO2 for 6 h. Cells were fixed at 6 hpi and then analyzed by IF. These data were acquired from at least two independent assays.

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  Fig. 10. Proposed docking model of PTC-209HBr with EV-A71 VP1 protein. (A) Molecular docking model of the binding between PTC-209HBr and EV-A71 capsid protein VP1. (B) The predicted amino acid residues may bind with PTC-209HBr or be related to resistance to TC-209HBr. The hydrogen bond is showed as a red dotted line, and some hydrophobic amino acids within 5 Å are labeled. The oxygen atom (red), nitrogen atom (blue) and sulfur atom (yellow) are also shown.

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  Fig. 11. The proposed model of PTC-209HBr inhibition of EV-A71. The model of the interaction between hSCARB2 and EV71 protomer as previously reported (Zhou et al., 2019). VP1, VP2, VP3, VP4 and hSCARB2 are coloured in blue, green, red, yellow and orange, respectively.