Enhanced human enterovirus 71 infection by endocytosis inhibitors reveals multiple entry pathways by enterovirus causing hand-foot-and-mouth diseases

Figures

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  Fig. 1. The effect of CPZ on EV71 infection in HepG2 and A549 cells. a. HepG2 and A549 cells were pretreated with increasing concentrations of CPZ (10, 20, 30 and 40 μM) for 2 h at 37 °C before EV71 infection. At 24 hpi, the infected cells were processed for flow cytometry. The bar charts represented the EV71 infectivity determined by the percentage of VP-1 positive cells and were shown as means with SD from three independent experiments. *, p < 0.05. Cell viability upon CPZ treatments was shown by the curve above the chart. b-d. A549 cells were infected with EV71 at an MOI of 5 for 12 h and then CPZ or DMSO was added. At 24 hpi, cells were lysed, and eIF4G cleavage, EV71- 2C, 3C and VP-1 were determined by Western blot (b); EV71 VP-1 mRNA levels were measured by qRT-PCR and normalized to 18 s (c); Virus titers were determined and shown in TCID50 (d). The blot shown was representative of three independent experiments and the full-length bolts were presented in Additional file 1 Fig. S5. Results were presented as the mean with SD from three independent experiments. *, p < 0.05. UT, untreated cells; TCID50, 50% tissue culture infective dose

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  Fig. 2. The effect of CPZ on EV71 infection is dynamin independent. a. Cells were infected by EV71 and treated with 20 μM CPZ or 80 μM DNS for 6 h (Vero, RD, MRC-5 and HEK293T, MOI = 1) or 12 h (Hela, A549, Huh7 and HepG2, MOI = 5). After removal of virus and the drugs, the cells were washed and continued to culture for 6 h or 12 h and then processed for flow cytometry. Means of three experiments with SD are shown. *, p < 0.05. b. A549 and RD cells were pretreated by 20 μM CPZ or 80 μM DNS for 2 h prior to incubation with AlexaFluor 488-conjugated transferrin (25 μg/ml) or AlexaFluor 594-conjugated cholera toxin B (CTxB, 10 μg/ml). After 15 min of incubation, cells were fixed and analyzed by immunofluorescent microscope. Arrow heads showed the bright puncta around nuclei. Scale bar, 100 μm. c. Various cells were infected by EV71 and treated by CPZ (20 μM), DNS (80 μM) or their combinations for 6 h (Vero, RD, MRC-5 and HEK293T, MOI = 1) or 12 h (Hela, A549, Huh7 and HepG2, MOI = 5). After removal of virus and the drug, cells were washed and continued to culture for 6 h or 12 h and then processed for flow cytometry. Means of three experiments with SD are shown. *, p < 0.05

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  Fig. 3. Characterization of the CPZ effect. a-b. HepG2 and A549 cells were infected by EV71 at an MOI of 5 in the presence of CPZ (20 μM) or DMSO for 6 h, 12 h and 18 h. After removal of virus and the drug, cells were washed and continued to culture until 24 h and then processed for flow cytometry. c-d. HepG2 and A549 cells were infected by EV71 at an MOI of 5. CPZ (20 μM) or DMSO was then added at −2, 0, 2, 4, 6, 8, 10, 12 hpi. At 24 hpi, cells were collected for VP-1 staining. The infectivity was measured as percentage of VP-1 positive cells. e-f. HepG2 and A549 cells were infected with EV71 at an MOI of 5 for 12 h, and then CPZ (20 μM) or DMSO was added. Cells were harvested at different time points (12 h, 16 h, 18 h and 24 h) afterward and subjected to VP-1 staining. Results were presented as the mean with SD from three independent experiments. *, p < 0.05. CPZ, filled square; DMSO, filled circle

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  Fig. 4. CPZ facilitates the entry step of EV71 infection. a. A549, RD and HepG2 cells were infected with EV71 pseudovirus or transfected with EV71 replicon RNA for 12 h before CPZ (20 μM) or DMSO treatment. Luciferase activity was quantified at 24 h post infection or transfection. The means with SD from three independent experiments each carried out in duplicate are shown. b. A549, RD and HepG2 cells were incubated with EV71 at an MOI of 50 and treated by CPZ or DMSO at 4 °C for 2 h. Then cells were washed extensively and lysed for viral RNA quantification with normalization to 18 s. Data shown are the means with SD from three independent experiments each carried out in duplicate. c. Immunofluorescence analysis was performed on RD and A549 cells infected with EV71 at an MOI of 10 (in RD) and 50 (in A549) in the presence or absence of 2.5 mM GnHCl. After 2 h incubation at 4 °C, cells were immediately shifted to 37 °C and treated by CPZ (20 μM) or DMSO. At 6 hpi, cells were washed extensively, and then fixed and stained with EV71 VP-1 antibody, and DAPI was used to visualize the nuclei. The images were one representative experiment out of three independent experiments. Arrow heads showed…

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  Fig. 5. The effect of CPZ on EV71 infection after depletion of CME. a. A549 and RD cells were transfected with vary siRNA or scramble siRNA for 96 h. Then cells were infected with EV71 at an MOI of 1 in the presence of 20 μM CPZ or DMSO for 24 h (A549) or 6 h (RD). Flow cytometry was applied to determine the viral infectivity. The representative percentages of the infected cells were indicated. b. The bar plots showed the summary of three independent experiments. The significance was determined by paired Student’s t test by comparing CPZ treatment to DMSO control (A549) or gene-knockdown group to scramble group (RD). *p < 0.05; **p < 0.01

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  Fig. 6. The effect of CPZ on CV-A16 infection. A549, Hela, RD and HepG2 cells were pretreated with CPZ (20 μM) or DMSO for 2 h. Then the cells were infected by CV-A16 at an MOI of 1. At 12 hpi (A549 and RD) or 24 hpi (HepG2 and Hela), flow cytometry was applied to determine the viral infectivity. The representative percentages of the infected cells were indicated (a). b. The plot showed the summary of three independent experiments. c. Virus titers in A549, RD, HepG2 and Hela cells were measured and shown in TCID50. The data were shown as means ± SD from three independent experiments. *, p < 0.05