Fig 6. Treatment with p38 MAPK inhibitor (SB203580) inhibits EV71 replication at the viral protein synthesis stage.
(A) Mock-infected RD cells were treated with SB203580 at different concentrations (10, 25, 50 and 100μM) or 1.0% DMSO (negative control) and cell lysates were harvested for Western blotting at 6h post-treatment. β-actin was included as a loading control. (B) Quantification of phospho-p38 MAPK (Thr180/Tyr182) and total p38 protein bands with reference to actin control bands (for each SB203580 concentration) and untreated control using ImageJ Gel Analysis program. (C) Cell viability of SB203580-treated cells and untreated control cells were measured using alamarBlue assay at 12h post-treatment. Values obtained were normalised against DMSO control. Virus titres in the supernatant of cells (denoted by bars) treated with varying concentrations of SB203580 post-adsorption were analysed via viral plaque assay. Error bars represent standard deviation (SD) of triplicate data. Statistical analyses were performed using one-way ANOVA and Dunnett’s test (Graphpad software) against untreated control **P < 0.01 (n = 3), *** P < 0.0001 (n = 3) versus 1.0% DMSO control. (D) RD cells were treated with 50uM SB203580 (p38 MAPK inhibitor) at different time points before and after infection in time-of-addition assay. Cell supernatants were harvested at 12hpi for quantification via viral plaque assays. Time-of-addition assay indicates that SB203580 acts between 2hpi and 10hpi to inhibit EV71 replication. In the co-treatment assay, SB203580 was added with the virus and no significant inhibition of EV71 infection was observed. Statistical analyses were performed using one-way ANOVA and Dunnett’s test (Graphpad software) against untreated control **P < 0.01 (n = 3), *** P < 0.0001 (n = 3) versus 0.5% DMSO control. (E) EV71 RNA synthesis was sensitive to SB203580 treatment. Quantitative RT-PCR assay revealed significant reduction in levels of EV71 RNA across increasing SB203580 concentration. Total RNA was extracted for all samples at 0, 8 and 10hpi and EV71 RNA levels were measured. CT values were normalised against actin and relative quantification of viral RNA level was determined. The ΔΔCt data were calculated from three independent experiments and error bars represent standard deviation for triplicate data sets. Fold difference of viral RNA for all samples was calculated relative to the RNA level in the DMSO control at 0hpi. Statistical analyses were carried out using one-way ANOVA with Dunnett’s test (Graphpad software). *P <0.05, **P <0.01 and *** P < 0.0001 (n = 3) vs the respective 1.0% DMSO control at each time-point. (F) Viral protein expression levels upon SB203580 treatment. EV71-infected RD cells were treated with SB203580 and cell lysates were harvested for Western blotting at 8h post-treatment. VP0 and VP2 viral protein expression was observed to decrease with increasing concentration of the p38 MAPK inhibitor. (G) Band intensities of VP0 and VP2 upon SB203580 treatment. The band intensities representing VP0 and VP2 protein expression level were quantitated with reference to actin control bands (for each concentration) and DMSO control using ImageJ Gel Analysis program. (H) Extracellular and intracellular virion levels upon p38 MAPK inhibition. Extracellular EV71 virions in the supernatant and intracellular virus particles were harvested separately at 12hpi for viral plaque assay to assess the effect of SB203580 treatment on virus packaging and release. p38 MAPK inhibition resulted in significant reduction in both intracellular and extracellular virions. Statistical analysis was performed using one-way ANOVA with Dunnett’s test (Graphpad software). **P <0.01, *** P < 0.0001 (n = 3) versus 1.0% DMSO control.