Limits...
The role of Misshapen NCK-related kinase (MINK), a novel Ste20 family kinase, in the IRES-mediated protein translation of human enterovirus 71.

Leong SY, Ong BK, Chu JJ - PLoS Pathog. (2015)

Bottom Line: We have also shown that viral RNA and protein expression level was significantly reduced upon MINK silencing, suggesting its involvement in viral protein synthesis which feeds into viral RNA replication process.Luciferase reporter assay further revealed that the translation efficiency of the EV71 internal ribosomal entry site (IRES) was reduced after blocking the MINK/p38 MAPK pathway.Further investigation on the effect of MINK silencing on heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) localisation demonstrated that cytoplasmic relocalisation of hnRNP A1 upon EV71 infection may be facilitated via the MINK/p38 MAPK pathway which then positively regulates the translation of viral RNA transcripts.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

ABSTRACT
Human Enterovirus 71 (EV71) commonly causes Hand, Foot and Mouth Disease in young children, and occasional occurrences of neurological complications can be fatal. In this study, a high-throughput cell-based screening on the serine/threonine kinase siRNA library was performed to identify potential antiviral agents against EV71 replication. Among the hits, Misshapen/NIKs-related kinase (MINK) was selected for detailed analysis due to its strong inhibitory profile and novelty. In the investigation of the stage at which MINK is involved in EV71 replication, virus RNA transfection in MINK siRNA-treated cells continued to cause virus inhibition despite bypassing the normal entry pathway, suggesting its involvement at the post-entry stage. We have also shown that viral RNA and protein expression level was significantly reduced upon MINK silencing, suggesting its involvement in viral protein synthesis which feeds into viral RNA replication process. Through proteomic analysis and infection inhibition assay, we found that the activation of MINK was triggered by early replication events, instead of the binding and entry of the virus. Proteomic analysis on the activation profile of p38 Mitogen-activated Protein Kinase (MAPK) indicated that the phosphorylation of p38 MAPK was stimulated by EV71 infection upon MINK activation. Luciferase reporter assay further revealed that the translation efficiency of the EV71 internal ribosomal entry site (IRES) was reduced after blocking the MINK/p38 MAPK pathway. Further investigation on the effect of MINK silencing on heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) localisation demonstrated that cytoplasmic relocalisation of hnRNP A1 upon EV71 infection may be facilitated via the MINK/p38 MAPK pathway which then positively regulates the translation of viral RNA transcripts. These novel findings hence suggest that MINK plays a functional role in the IRES-mediated translation of EV71 viral RNA and may provide a potential target for the development of specific antiviral strategies against EV71 infection.

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4352056&req=5

ppat.1004686.g006: 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.

Mentions: To ascertain the role of p38 MAPK signalling pathway in EV71 replication, the p38 MAPK inhibitor SB203580 [30] was examined for its impact on EV71 replication. To verify the efficacy of SB203580 in inhibiting the p38 MAPK pathway, we measured the phosphorylation levels of p38 MAPK via Western blot analysis. A dose-dependent reduction in the phosphorylation of p38 MAPK was observed with increasing concentration of SB203580 and significant inhibition was noticed at a concentration of 50μM (Fig. 6A and 6B). Consistent expression levels of total p38 MAPK at different concentrations of SB203580 suggested that SB203580 inhibited p38 MAPK phosphorylation but not its expression.


The role of Misshapen NCK-related kinase (MINK), a novel Ste20 family kinase, in the IRES-mediated protein translation of human enterovirus 71.

Leong SY, Ong BK, Chu JJ - PLoS Pathog. (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352056&req=5

ppat.1004686.g006: 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.
Mentions: To ascertain the role of p38 MAPK signalling pathway in EV71 replication, the p38 MAPK inhibitor SB203580 [30] was examined for its impact on EV71 replication. To verify the efficacy of SB203580 in inhibiting the p38 MAPK pathway, we measured the phosphorylation levels of p38 MAPK via Western blot analysis. A dose-dependent reduction in the phosphorylation of p38 MAPK was observed with increasing concentration of SB203580 and significant inhibition was noticed at a concentration of 50μM (Fig. 6A and 6B). Consistent expression levels of total p38 MAPK at different concentrations of SB203580 suggested that SB203580 inhibited p38 MAPK phosphorylation but not its expression.

Bottom Line: We have also shown that viral RNA and protein expression level was significantly reduced upon MINK silencing, suggesting its involvement in viral protein synthesis which feeds into viral RNA replication process.Luciferase reporter assay further revealed that the translation efficiency of the EV71 internal ribosomal entry site (IRES) was reduced after blocking the MINK/p38 MAPK pathway.Further investigation on the effect of MINK silencing on heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) localisation demonstrated that cytoplasmic relocalisation of hnRNP A1 upon EV71 infection may be facilitated via the MINK/p38 MAPK pathway which then positively regulates the translation of viral RNA transcripts.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

ABSTRACT
Human Enterovirus 71 (EV71) commonly causes Hand, Foot and Mouth Disease in young children, and occasional occurrences of neurological complications can be fatal. In this study, a high-throughput cell-based screening on the serine/threonine kinase siRNA library was performed to identify potential antiviral agents against EV71 replication. Among the hits, Misshapen/NIKs-related kinase (MINK) was selected for detailed analysis due to its strong inhibitory profile and novelty. In the investigation of the stage at which MINK is involved in EV71 replication, virus RNA transfection in MINK siRNA-treated cells continued to cause virus inhibition despite bypassing the normal entry pathway, suggesting its involvement at the post-entry stage. We have also shown that viral RNA and protein expression level was significantly reduced upon MINK silencing, suggesting its involvement in viral protein synthesis which feeds into viral RNA replication process. Through proteomic analysis and infection inhibition assay, we found that the activation of MINK was triggered by early replication events, instead of the binding and entry of the virus. Proteomic analysis on the activation profile of p38 Mitogen-activated Protein Kinase (MAPK) indicated that the phosphorylation of p38 MAPK was stimulated by EV71 infection upon MINK activation. Luciferase reporter assay further revealed that the translation efficiency of the EV71 internal ribosomal entry site (IRES) was reduced after blocking the MINK/p38 MAPK pathway. Further investigation on the effect of MINK silencing on heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) localisation demonstrated that cytoplasmic relocalisation of hnRNP A1 upon EV71 infection may be facilitated via the MINK/p38 MAPK pathway which then positively regulates the translation of viral RNA transcripts. These novel findings hence suggest that MINK plays a functional role in the IRES-mediated translation of EV71 viral RNA and may provide a potential target for the development of specific antiviral strategies against EV71 infection.

No MeSH data available.


Related in: MedlinePlus