Fig 1.
2DE-gel electrophoresis of EV71-infected NSC-34 cells.
(a) NSC-34 cells were infected with EV71 at M.O.I. 10 and cell lysate was prepared at 6, 24, 48 and 72 h.p.i. for 2DE gel electrophoresis. Representative 2DE image of infected cell lysate at 48 h.p.i. is shown. Three independent experiments were performed. UI, uninfected. (b) Heat map was generated using MultiExperiment Viewer (MeV). Distance was represented by Euclidean average linkage clustering. The host proteins are further categorized into (c) heavily up-regulated and (d) down-regulated proteins.
Fig 2.
The protein network analysis was performed using STRING v10. The confidence level of protein interactions is indicated by the thickness of connecting line. The interactions network is significantly enriched (p<0.05). The protein symbols used in this network analysis are listed in S2 Table.
Fig 3.
siRNA-mediated gene silencing in NSC-34 cells.
NSC-34 cells were transfected with various concentrations of ON-TARGET PLUS siRNA SMARTpools. (a) At 48 h.p.t., the cell viability of transfected cells was assessed using alamarBlue assay. (b) Knocked-down NSC-34 cells were infected with EV71 at M.O.I. 10. The viral titers in the culture supernatant were determined by plaque assay at 48 h.p.i.. Statistical analysis was performed using one-way ANOVA test with Dunnett’s posttest (* p<0.1, ** p<0.01, *** p<0.001, **** p<0.0001). (c) NSC-34 cells were transfected with PHB siRNA pool or with non-targeting siRNA (NTC) control at various concentrations. The efficiency of siRNA knockdown was verified at 48 h.p.t. by Western blot. (d) PHB siRNA- or siNTC-transfected cells were infected with EV71 at M.O.I. 10. The viral titers in the culture supernatants were determined by plaque assay at 48 h.p.i.. Cell viability of the transfected cells was assessed using alamarBlue assay. Statistical analysis was performed using two-tailed student’s t-test (** p<0.005, *** p<0.005). Relative band quantification (below Western blot) was determined by ImageJ, by normalizing to loading control, β-actin. Error bars represent mean ± standard deviation. One representative of two biological repeats is shown.
Fig 4.
Involvement of PHB in virus entry.
(a) Blocking antibody assay. NSC-34 cells were pre-treated with anti-PHB or IgG isotype antibodies, followed by EV71 infection at M.O.I. 10. Culture supernatants were harvested at 48 h.p.i. for viral titer determination by plaque assay. Cell viability was assessed by alamarBlue assay. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test (***, p<0.0005; ****, p<0.0001). A representative experiment of two independent repeats is shown. (b) Proximity ligation assay. NSC-34 (M.O.I. 20) and RD cells (M.O.I. 5) were incubated with EV71 at 4°C for 2 hours to allow viral adsorption, prior to PLA staining. SCARB2-stained NSC-34 and RD cells served as negative and positive controls, respectively. Scale bar represents 20μm. A representative experiment of two independent repeats is shown. (c) Co-immunoprecipitation of EV71 and surface-expressed PHB. EV71 and NSC-34 cells were co-incubated for 2 hours at 4°C. The cell lysate was pulled down with anti-PHB or IgG isotype control antibodies prior to immunoblotting using anti-VP1 primary antibody. Mock-infected cells and IgG isotype served as control. A representative experiment of two independent repeats is shown. IP, immunoprecipitation; IB, immunoblot.
Fig 5.
Involvement of intracellular PHB in viral genome replication.
(a) NSC-34 cells were reverse-transfected with 50 nM of PHB siRNA, followed by transfection with EV71 purified RNA genome. Culture supernatant was harvested at the indicated time points post-transfection for viral titer determination by plaque assay. Statistical analysis was performed using two-way ANOVA test with Sidak’s multiple comparisons test (****, p< 0.0001). Viral RNA only transfection served as control. A representative experiment is shown from two independent repeats. (b) Schematic drawing of lucEV71 replicon. (c) NSC-34 cells were reverse-transfected with 50 nM of PHB siRNA, siNTC or left untreated prior to transfection with 1 μg of lucEV71 RNA. Luminescence signal was read at 48 h.p.t. Statistical analysis was performed using one-way ANOVA with Dunnet’s post-test (****, p<0.0001). Error bars represent mean ± standard deviation. One representative of two biological repeats is shown.
Fig 6.
Intracellular PHB co-localizes with EV71 replication complex.
(a) NSC-34 cells were infected with EV71 at M.O.I. 10 and fixed with methanol at 48 h.p.i. prior to immunostaining. Uninfected cells served as control. Scale bar represents 20μm. (b) Total cell lysate of infected NSC-34 cells was pulled down with anti-PHB or IgG isotype control antibodies prior to immunoblotting. Mock-infected cells and IgG isotype served as control. IP, immunoprecipitation; IB, immunoblot. One representative of two independent experiments is shown.
Fig 7.
Involvement of mitochondrial PHB in viral replication.
(a-c) NSC-34 cells were seeded onto coverslips and infected with EV71 at M.O.I. 10. At 48 h.p.i., the cells were fixed using ice cold methanol and subjected to immunostaining using specific antibodies. Images were post-processed using ImageJ to reveal the co-localization signal. Scale bar represents 20 μm. (d) NSC-34 and RD cells were infected with EV71 at M.O.I. 10 and 1, respectively. At 48 h.p.i. (NSC-34 cells) and 12 h.p.i. (RD cells), the cells were lysed, and the mitochondrial fraction was subjected to Western blot analysis. UI, uninfected; INF, EV71-infected. (e) TEM of EV71-infected NSC-34 cells. NSC-34 cells were infected with EV71 at M.O.I 20 for 48 hours and processed for TEM analysis. M, mitochondria; Nuc, nucleus; V, virus. One representative of two independent experiments is shown.
Fig 8.
Roc-A treatment of EV71-infected NSC-34 cells.
NSC-34 cells were first infected with EV71 at M.O.I. 10, followed by Roc-A treatment for 48 hours. At 48 h.p.i. the culture supernatant was collected for viral titer determination by plaque assay (a). Furthermore, the cell lysate was prepared for Western blot analysis (b). Relative band quantification (below Western blot) was determined by ImageJ, by normalizing to loading control, β-actin. Cell viability was assessed using alamarBlue assay. (c) Immunostaining of Roc-A treated NSC-34 cells. At 48 hours incubation the cells were fixed with ice cold methanol and probed with specific antibodies. Scale bar denotes 20 μm. (d) Mitotoxicity of Roc-A in NSC-34 cells. Cells were incubated with various concentrations of Roc-A for 48 hours before assessment of cytotoxicity (fluorescence) and mitotoxicity (luminescence) using Mitochondrial ToxGlo Assay. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test (*, p<0.05; **, p<0.005; ***, p<0.001; ****, p<0.0001). One representative from two independent experiments is shown.
Fig 9.
Role of PHB in EV71-infected SK-N-SH cells.
(a) SK-N-SH cells were transfected with PHB siRNA at various concentrations for 48 hours. The efficiency of siRNA knockdown was verified by Western blot. PHB-knockdown SK-N-SH cells were infected with EV71 at M.O.I. 1. Viral titers in the culture supernatant were determined by plaque assay at 48 h.p.i. Statistical analysis was performed using two-tailed student’s t-test (** p<0.005, *** p<0.005). Non-targeting siRNA (NTC) served as control. (b) SK-N-SH cells were pre-treated with anti-PHB antibodies followed by EV71 infection at M.O.I. 1. Culture supernatant was harvested at 48 h.p.i. for viral titer determination by plaque assay. IgG isotype antibodies served as control. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test (***, p<0.0005; ****, p<0.0001). (c) SK-N-SH cells were reverse-transfected with 50 nM PHB siRNA, siNTC or left untreated prior to transfection with 0.25 μg of lucEV71 RNA. Luminescence signal was read at 48 h.p.t. Statistical analysis was performed using one-way ANOVA with Dunnet’s post test (**, p<0.005). (d) SK-N-SH cells were first infected with EV71 at M.O.I. 1 followed by Roc-A treatment for 48 hours. At 48 h.p.i. the culture supernatant was collected for viral titer determination by plaque assay, and the cell lysate was harvested for Western blot analysis. Statistical analysis was performed using one-way ANOVA with Dunnett’s post-test (**, p<0.005). Relative band quantification (below Western blot) was determined by ImageJ, by normalizing to loading control, β-actin. Error bars represent mean ± standard deviation. Cellular cytotoxicity was assessed using alamarBlue assay. One representative from two independent experiments is shown.
Fig 10.
PHB expression and Roc-A treatment in EV71-infected AG129 mice.
(a) Two week-old AG129 mice (n = 3) were infected i.p. with EV71 (107 PFU) and the limb muscles, spinal cord and brainstem were harvested for immunohistochemical analysis at day 4 p.i. Scale bars denote 100 μm (20× magnification) and 50 μm (40× magnification). (b) EV71-infected AG129 mice (n = 8) were treated i.p. with Roc-A at 0.25 mg/kg at day 1 and 3 p.i. and were monitored for survival and clinical manifestations. Clinical scores were defined as follows: 0, healthy; 1, ruffled hair and hunched back; 2, limb weakness; 3, one limb paralysis; 4, both limbs paralysis at which point the animals were euthanized. Statistical analysis of survival curve was performed using logrank (Mantel-Cox) test (**, p<0.005). (c) Limb muscles, spinal cord and brain were harvested at day 4 p.i. for viral load determination by plaque assay (n = 6/7). Dotted line represents the limit of detection. Statistical analysis was performed using Mann-Whitney U test (*, p<0.05). Error bars represent mean ± SEM. One representative of two biological repeats is shown.