Fig 1.
Human serine/threonine kinase siRNA library screen.
Effect of gene knockdown by siRNA on EV71 replication analysed from primary screen. 40% reduction in viral antigen positive cells was considered as the acceptable level of virus inhibition and positive hits are genes which resulted in a percentage of viral antigen positive cells of less than 60% upon the knockdown of these genes. As such, 6 genes have been identified as positive hits from the primary screen. First 6 bars represent siRNA controls used while the other bars represent the host serine/threonine kinases targeted in the screening. siRNA controls utilised included non-targeting siRNAs as well as siRNAs targeting several housekeeping genes. Values obtained in the graph were normalised against the mean of the transfection control (EV71-infected cells treated with only the transfection reagent).
Fig 2.
Silencing of MINK significantly reduced EV71 replication in a siRNA concentration-dependent manner.
(A) siRNA-treated EV71 infected cells were fixed at the same time-points and intracellular viruses were detected by immunofluorescence assay. Immunofluorescence detection of EV71 VP2 proteins (green) with the nuclei stained with DAPI (blue) is shown. The images were taken at 10X magnification. Cells in both the negative and transfection controls were not infected with EV71 while cells in the positive control were infected with EV71 in the absence of siRNA. (B) Cell viability of siRNA-treated cells was measured in relation to untreated cells using alamarBlue assay after 72h incubation. Virus titres in the supernatant of siRNA-treated cells were analysed via viral plaque assay. Error bars represent standard deviation (SD) of triplicate data and values obtained were normalised against the transfection control. Statistical analyses were performed using one-way ANOVA and Dunnett’s test (Graphpad software) against untreated control. *P <0.05 (n = 3), **P <0.01 (n = 3). (C) Verification of gene knockdown efficiency of MINK siRNA SMARTpool at concentrations ranging from 0nM to 45nM. Western blot analysis was performed to detect protein expression levels of MINK, with β-actin as the loading control. Parallel transfection of scrambled siRNA served as a knockdown control. MINK protein expression was observed to decrease in a dose-dependent manner across siRNA concentration. (D) Band intensity of MINK gene knockdown verification. The band intensities representing MINK protein expression level were quantitated with reference to actin control bands (for each individual concentration) and PTC. The intensities of protein bands were quantitated using ImageJ Gel Analysis program. (E) Verification of gene knockdown efficiency of individual siRNA within the siRNA SMARTpool directed against MINK at 45nM. Western blot analysis was performed to detect protein expression levels of MINK, with β-actin as the loading control. (F) Band intensity of MINK gene knockdown verification in deconvolution assay. The band intensities representing MINK protein expression level were quantitated with reference to actin control bands (for each individual siRNA) and PTC. (G) Virus titres in the supernatant of cells treated with individual siRNAs within siRNA SMARTpool 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 <0.0001 (n = 3). (H) Virus titres of other human enteroviruses (Echovirus 7, Coxsackievirus A6 and EV71 strain 41) in the supernatant of siRNA-treated cells 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 scrambled control (Scr). *P < 0.05, **P < 0.01 and ***P < 0.0001 (n = 3) versus scrambled control.
Fig 3.
MINK plays an essential role in EV71 viral protein synthesis.
(A) EV71 viral RNA was transfected into RD cells pre-treated with MINK siRNA and supernatant was harvested from cells at 12h post-infection (hpi) for viral plaque assay. Silencing of MINK with targeting siRNA continued to cause inhibition of virus replication. Statistical analysis was performed using one-way ANOVA with Dunnett’s test (Graphpad software). *** P < 0.0001 (n = 3) versus untreated control (0nM). (B) EV71 RNA synthesis was sensitive to silencing efficiencies of MINK. Quantitative RT-PCR assay revealed significant reduction in levels of EV71 RNA across increasing siRNA concentration in MINK siRNA-treated cells. 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 transfection control (PTC) at 0hpi. Statistical analyses were carried out using one-way ANOVA with Dunnett’s test (Graphpad software). *P<0.05 and *** P < 0.0001 (n = 3) vs the respective PTC at each time-point. (C) Time course study of EV71 structural protein expression via Western blot analysis. Upper band (36kDa) represents VP0 while lower band (28 kDa) represents VP2. β-actin was used as the loading control. (D) Band intensity of VP0 and VP2 in time course study. The band intensities representing VP0 and VP2 protein expression level were quantitated with reference to actin control bands (for each time-point) and 0hpi using ImageJ Gel Analysis program. (E) Viral protein expression levels upon the silencing of MINK. VP0 and VP2 viral protein expression was observed to decrease with increasing concentration of siRNA targeting MINK. (F) Band intensities of VP0 and VP2 upon siRNA knockdown of MINK. The band intensities representing VP0 and VP2 protein expression level were quantitated with reference to actin control bands (for each siRNA concentration) and 0nM using ImageJ Gel Analysis program. (G) Extracellular and Intracellular virion levels upon the silencing of MINK. Extracellular EV71 virions in the supernatant and intracellular virus particles were harvested separately at 12hpi for viral plaque assay to assess the effect of siRNA knockdown of MINK on virus packaging and release. Silencing of MINK 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 and *** P < 0.0001 (n = 3) versus untreated control (0nM).
Fig 4.
Phosphorylation of MINK is triggered post-entry by early replication events.
Phos-tag acrylamide binds phosphorylated proteins and retards their migration to separate the phosphorylated proteins from their unphosphorylated counterparts. Total MINK antibody was used to detect both phosphorylated (upper bands) and unphosphorylated MINK (lower bands). β-actin was used as a loading control. (A) Viral RNA was transfected into cells and cell lysates were harvested at indicated time-points to assess the phospho-MINK levels. Phospho-MINK levels in RNA-transfected cells were comparable to the infection control at the same time-points. (B) The band intensities representing MINK phosphorylation level were quantitated with reference to actin control bands (for each time-point) and 0h using ImageJ Gel Analysis program. (C) Virus titres in the supernatant of cells treated with the anti-SCARB2 and anti-IgG antibodies were analysed via viral plaque assay. Blocking SCARB2 receptors with increasing concentration of SCARB2 antibody resulted in a significant reduction in virus titres. 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 <0.0001 (n = 3) (D) Blocking SCARB2 receptors with increasing concentration of SCARB2 antibody did not affect the phosphorylation of MINK in cells at 6h after addition of virus. (E) The band intensities representing MINK phosphorylation level were quantitated with reference to actin control bands (for each concentration) and 0μg/mL using ImageJ Gel Analysis program.
Fig 5.
EV71 infection triggers p38 MAPK phosphorylation downstream of MINK.
Western blot analysis was performed to assess the levels of phosphorylated p38 MAPK (phospho-p38) at 0, 2, 4, 6, 8, 10 and 12hpi. Total p38 (t-p38) was probed as an internal control for p38 MAPK protein expression and β-actin was used as a loading control. (A) Infection with infectious EV71 was observed to activate p38 MAPK phosphorylation from 6hpi and was most significant at 8hpi. (B) Phosphorylation levels of p38 MAPK in mock-infected cells was basal and constant across the 12h. (C) Cells exposed to UV-inactivated EV71 virus also displayed basal and constant level of p38 MAPK phosphorylation of p38 MAPK. (D) Quantification of phospho-p38 MAPK (Thr180/Tyr182) protein bands. The band intensities representing phospho-p38 MAPK level were quantitated with reference to actin control bands (for each time-point) and 0hpi using ImageJ Gel Analysis program. (E) Western blot analysis of the phosphorylation levels of p38 MAPK at 8hpi in siRNA-treated cells. The left panel shows the phospho-p38 levels in EV71-infected under three different treatments: no treatment, scrambled siRNA treatment and MINK targeting siRNA treatment. The right panel shows the phospho-p38 levels in mock-infected cells under the same treatments. (F) Quantification of MINK protein bands with reference to actin control bands (for each concentration) and PTC using ImageJ Gel Analysis program. (G) Quantification of phospho-p38 MAPK (Thr180/Tyr182) and total p38 protein bands with reference to actin control bands (for each concentration) and PTC using ImageJ Gel Analysis program.
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.
Fig 7.
Silencing of MINK and inhibition of p38 MAPK phosphorylation reduces translation efficiency of EV71 IRES.
(A) Schematic diagram of the bicistronic construct containing the Renilla luciferase (RLuc) and firefly luciferase (FLuc) genes controlled by the cytomegalovirus (CMV) promoter and 5’ UTR of EV71–26M [67], respectively. (B) Effect of knockdown of MINK on EV71 IRES activity. RD cells were pre-treated with MINK or scrambled siRNA. Three days after transfection, the bicistronic construct was then transfected into the cells. Luciferase activity was measured 24h after transfection. Amantadine, an inhibitor of EV71 IRES [33], was added to untreated cells to serve as negative control for IRES activity. Untreated cells that were transfected with the bicistronic construct were used as positive control. The FLuc/RLuc ratio for each sample were normalised to the FLuc/RLuc ratio of untreated control. Dose-dependent reduction in relative translation efficiency of the IRES was observed in MINK siRNA-treated cells. Error bars represent standard deviation of triplicate data sets. Statistical analyses were performed using one-way ANOVA with Dunnett’s test (Graphpad software). *P < 0.05, **P < 0.01 and *** P < 0.0001 versus untreated control. (C) Effect of p38 MAPK inhibition on EV71 IRES activity. Relative translation efficiency was determined as the ratio of FLuc to RLuc for each sample and the FLuc/ RLuc ratio for each sample were normalised to the FLuc/RLuc ratio of DMSO control, expressed as percentage. Error bars reflect the standard deviation of triplicate data sets. Transfected cells with the bicistronic construct without drug treatment (DMSO control) was used as positive control. Error bars represent standard deviation of triplicate data sets. Statistical analyses were performed using one-way ANOVA with Dunnett’s test (Graphpad software). *P < 0.05, **P < 0.01 and *** P < 0.0001 versus 1.0% DMSO control.
Fig 8.
MINK silencing and p38 MAPK inhibition in EV71-infected cells inhibits cytoplasmic localisation of hnRNP A1.
(A) RD cells were pre-treated with MINK targeting and scrambled siRNA and subjected to infection with EV71. siRNA-treated cells were fixed and the subcellular localisation of hnRNP A1 (red), an IRES-transacting factor, was investigated by indirect immunofluorescence assay. Immunofluorescence detection of double-stranded RNA (dsRNA, green) with the nuclei stained with DAPI (blue) was shown to indicate EV71 infection. The images were taken at 100X magnification. Colocalisation quantification was based on the Manders Overlap Coefficient (MOC) using whole-cell immunofluorescence (WCIF) ImageJ software [36] and represented as percent colocalisation at the respective siRNA concentrations. Error bars represent the standard deviation of duplicate data. (B) RD cells were subjected to infection with EV71 and post-treated with SB203580 (p38 MAPK inhibitor) for 8h. SB203580-treated cells were fixed and the subcellular localisation of hnRNP A1 (red) was investigated by indirect immunofluorescence assay. Mock-infected and DMSO-treated cells were included as infection and solvent control, respectively. The images were taken at 100X magnification. Colocalisation quantification was based on the MOC using WCIF ImageJ software and represented as percent colocalisation at the respective drug concentrations. Error bars represent the standard deviation of duplicate data.
Fig 9.
Proposed mechanism of action of MINK in the EV71 replication cycle.
EV71 infection stimulates MINK activation which in turn triggers the phosphorylation of p38 MAPK downstream. The phosphorylation of p38 MAPK triggers a kinase cascade which results in the cytoplasmic relocalisation of hnRNP A1. hnRNP A1 binds to the viral IRES and promotes the recruitment of ribosomes at the IRES at the 5’ untranslated region (UTR) of EV71 genome, stimulating the IRES-mediated viral protein translation.