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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

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.
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ppat.1004686.g004: 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.

Mentions: As a MAP kinase kinase kinase kinase (MAP4K), MINK is activated upstream in MAPK pathways and thus we hypothesised that the early events in EV71 infection could be responsible for the activation of MINK. To investigate if virus binding and entry triggered the phosphorylation of MINK, Western blot analysis on phospho-MINK was conducted after the transfection of viral RNA into cells to bypass the normal entry processes of EV71. Since phospho-MINK antibodies are not available commercially, a phosphate-binding tag (Phos-Tag) [27] was used to separate the phosphorylated proteins from the unphosphorylated proteins. 6h and 8h were selected as the time-points for harvest of cell lysates due to the significant increase in phospho-MINK levels at these time-points after infection (S1 Fig). As shown in Fig. 4A and Fig. 4B, cells transfected with viral RNA displayed similar phospho-MINK levels at 6h and 8h as the infection control (EV71-infected), suggesting that initial binding and entry processes of the virus was not required for the activation of MINK upon EV71 infection.


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)

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.
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ppat.1004686.g004: 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.
Mentions: As a MAP kinase kinase kinase kinase (MAP4K), MINK is activated upstream in MAPK pathways and thus we hypothesised that the early events in EV71 infection could be responsible for the activation of MINK. To investigate if virus binding and entry triggered the phosphorylation of MINK, Western blot analysis on phospho-MINK was conducted after the transfection of viral RNA into cells to bypass the normal entry processes of EV71. Since phospho-MINK antibodies are not available commercially, a phosphate-binding tag (Phos-Tag) [27] was used to separate the phosphorylated proteins from the unphosphorylated proteins. 6h and 8h were selected as the time-points for harvest of cell lysates due to the significant increase in phospho-MINK levels at these time-points after infection (S1 Fig). As shown in Fig. 4A and Fig. 4B, cells transfected with viral RNA displayed similar phospho-MINK levels at 6h and 8h as the infection control (EV71-infected), suggesting that initial binding and entry processes of the virus was not required for the activation of MINK upon EV71 infection.

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