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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 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.
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ppat.1004686.g008: 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.

Mentions: Since eIF4E was unlikely to be the effector of the MINK/p38 MAPK pathway in our study, we hypothesized that the MINK/p38 MAPK signalling pathway might activate an IRES tran-sacting factor (ITAF) downstream, which thus resulted in the positive regulation of the EV71 IRES translation efficiency. Heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) is predominantly a nuclear protein but shuttles back and forth between the nucleus and cytoplasm. It has been reported that hnRNP A1 act as an ITAF that relocalises in the cytoplasm where it interacts with the EV71 IRES upon infection, promoting its translation efficiency [20]. As previous study has also shown that p38 MAPK signalling is implicated in the cytoplasmic accumulation of hnRNP A1 in uninfected cells [35], we were interested to know whether MINK/p38 MAPK signalling modulate the EV71 IRES activity by altering the subcellular localisation of hnRNP A1. Fig. 8A shows the immunofluorescence staining of hnRNP A1 in EV71-infected cells at 8h post-infection. The degree of colocalisation between the hnRNP A1 protein (stained with rhodamine) and the cell nucleus (stained with DAPI) was quantified using Manders coefficient [36]. The level of the hnRNP A1 in the nucleus increased significantly upon the siRNA-knockdown of MINK (74.8% colocalisation, Fig. 8A xiii-xvi) compared to the scrambled control (37.2% colocalisation, Fig. 8A ix-xii), resembling the state of hnRNP A1 in mock-infected cells (80.1% colocalisation, Fig. 8A i-iv). These data demonstrated that the silencing of MINK reduced the hnRNP A1 signals in the cytoplasm as the degree of colocalisation between the hnRNP A1 signals and DAPI signals in the nucleus increased upon the siRNA knockdown of MINK.


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 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.
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Related In: Results  -  Collection

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ppat.1004686.g008: 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.
Mentions: Since eIF4E was unlikely to be the effector of the MINK/p38 MAPK pathway in our study, we hypothesized that the MINK/p38 MAPK signalling pathway might activate an IRES tran-sacting factor (ITAF) downstream, which thus resulted in the positive regulation of the EV71 IRES translation efficiency. Heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) is predominantly a nuclear protein but shuttles back and forth between the nucleus and cytoplasm. It has been reported that hnRNP A1 act as an ITAF that relocalises in the cytoplasm where it interacts with the EV71 IRES upon infection, promoting its translation efficiency [20]. As previous study has also shown that p38 MAPK signalling is implicated in the cytoplasmic accumulation of hnRNP A1 in uninfected cells [35], we were interested to know whether MINK/p38 MAPK signalling modulate the EV71 IRES activity by altering the subcellular localisation of hnRNP A1. Fig. 8A shows the immunofluorescence staining of hnRNP A1 in EV71-infected cells at 8h post-infection. The degree of colocalisation between the hnRNP A1 protein (stained with rhodamine) and the cell nucleus (stained with DAPI) was quantified using Manders coefficient [36]. The level of the hnRNP A1 in the nucleus increased significantly upon the siRNA-knockdown of MINK (74.8% colocalisation, Fig. 8A xiii-xvi) compared to the scrambled control (37.2% colocalisation, Fig. 8A ix-xii), resembling the state of hnRNP A1 in mock-infected cells (80.1% colocalisation, Fig. 8A i-iv). These data demonstrated that the silencing of MINK reduced the hnRNP A1 signals in the cytoplasm as the degree of colocalisation between the hnRNP A1 signals and DAPI signals in the nucleus increased upon the siRNA knockdown of MINK.

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