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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

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).
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ppat.1004686.g003: 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).

Mentions: Further experiments were performed to elucidate the involvement of MINK within the different stages of the EV71 replication processes (viral entry, viral RNA replication and viral protein synthesis). To assess the involvement of MINK in viral entry, viral RNA was extracted and transfected into cells which were pre-treated with MINK siRNA to bypass the normal viral entry processes ie. clathrin-mediated endocytosis for EV71 [26]. As such, infectious virus titre obtained from viral plaque assays would assist in the elucidation of the potential involvement of MINK in the viral entry stage. In this assay, a dose-dependent reduction in the virus yield was observed across the siRNA concentrations with a maximum reduction of ~1.8 log at 45nM (Fig. 3A), indicating that the silencing of MINK continued to cause virus inhibition despite bypassing the viral entry stage. This result suggested that MINK might play a more essential role at the post-entry stage.


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)

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).
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ppat.1004686.g003: 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).
Mentions: Further experiments were performed to elucidate the involvement of MINK within the different stages of the EV71 replication processes (viral entry, viral RNA replication and viral protein synthesis). To assess the involvement of MINK in viral entry, viral RNA was extracted and transfected into cells which were pre-treated with MINK siRNA to bypass the normal viral entry processes ie. clathrin-mediated endocytosis for EV71 [26]. As such, infectious virus titre obtained from viral plaque assays would assist in the elucidation of the potential involvement of MINK in the viral entry stage. In this assay, a dose-dependent reduction in the virus yield was observed across the siRNA concentrations with a maximum reduction of ~1.8 log at 45nM (Fig. 3A), indicating that the silencing of MINK continued to cause virus inhibition despite bypassing the viral entry stage. This result suggested that MINK might play a more essential role at the post-entry stage.

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