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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

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.
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ppat.1004686.g005: 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.

Mentions: After determining the triggering event of MINK upon EV71 infection, we next investigated the mechanism of action of MINK on EV71 viral protein synthesis. It has been reported that MINK activates the p38 MAPK pathway [23], a signalling pathway that has also been shown to play a role in the replication of Encephalomyelitis virus (EMCV), a member of the Picornaviridae family [25]. As such, we examined the activation profile of p38 MAPK upon EV71 infection to assess whether the p38 MAPK signalling pathway is activated during EV71 replication. As serum has also been reported to induce phosphorylation of certain proteins [29], fecal calf serum (FCS) was removed from the virus stock and growth media in the course of this experiment to reduce the additional activation of p38 MAPK by the serum. Cell lysates were analysed at indicated time-points post-infection for 12h by Western blotting to examine the changes in the phosphorylation levels of p38 MAPK (phospho-p38). Constant and basal phosphorylation of p38 MAPK was observed in mock-infected cells throughout the 12h time course (Fig. 5B and 5D). In contrast, the EV71-infected cells showed an increase in the phosphorylation level of p38 MAPK between 6 to 8h post-infection (Fig. 5A and 5D), followed by a subsequent decrease from 8 to 12h post-infection. To demonstrate the dependency of p38 MAPK phosphorylation on EV71 replication, we also examined the phospho-p38 MAPK profile in RD cells infected with UV-inactivated EV71 (Fig. 5C and 5D). Similar to the mock-infected control, cells exposed to UV-inactivated EV71 showed constant phosphorylation level of p38 MAPK throughout the 12h time course, indicating that attachment of the virions to cell surface receptors or virus entry process were not sufficient to trigger the phosphorylation of p38 MAPK. Total p38 MAPK was also assessed to ensure that the changes in phospho-p38 MAPK levels were not due to differences in p38 MAPK expression levels. Phospho-p38 MAPK levels at 0h post-infection appears higher in Fig. 5A in EV71-infected samples than that in the mock-infected samples (Fig. 5B) and samples exposed to UV-inactivated EV71 (Fig. 5C) probably due to more total proteins loaded as seen from the total p38 and β-actin levels. However, it is evident in the trend of p38 MAPK activation profile that phospho-p38 MAPK levels were significantly increased upon EV71 infection (Fig. 5D). Hence, these results suggested that activation of p38 MAPK signalling pathway requires the active replication of EV71.


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)

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.
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ppat.1004686.g005: 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.
Mentions: After determining the triggering event of MINK upon EV71 infection, we next investigated the mechanism of action of MINK on EV71 viral protein synthesis. It has been reported that MINK activates the p38 MAPK pathway [23], a signalling pathway that has also been shown to play a role in the replication of Encephalomyelitis virus (EMCV), a member of the Picornaviridae family [25]. As such, we examined the activation profile of p38 MAPK upon EV71 infection to assess whether the p38 MAPK signalling pathway is activated during EV71 replication. As serum has also been reported to induce phosphorylation of certain proteins [29], fecal calf serum (FCS) was removed from the virus stock and growth media in the course of this experiment to reduce the additional activation of p38 MAPK by the serum. Cell lysates were analysed at indicated time-points post-infection for 12h by Western blotting to examine the changes in the phosphorylation levels of p38 MAPK (phospho-p38). Constant and basal phosphorylation of p38 MAPK was observed in mock-infected cells throughout the 12h time course (Fig. 5B and 5D). In contrast, the EV71-infected cells showed an increase in the phosphorylation level of p38 MAPK between 6 to 8h post-infection (Fig. 5A and 5D), followed by a subsequent decrease from 8 to 12h post-infection. To demonstrate the dependency of p38 MAPK phosphorylation on EV71 replication, we also examined the phospho-p38 MAPK profile in RD cells infected with UV-inactivated EV71 (Fig. 5C and 5D). Similar to the mock-infected control, cells exposed to UV-inactivated EV71 showed constant phosphorylation level of p38 MAPK throughout the 12h time course, indicating that attachment of the virions to cell surface receptors or virus entry process were not sufficient to trigger the phosphorylation of p38 MAPK. Total p38 MAPK was also assessed to ensure that the changes in phospho-p38 MAPK levels were not due to differences in p38 MAPK expression levels. Phospho-p38 MAPK levels at 0h post-infection appears higher in Fig. 5A in EV71-infected samples than that in the mock-infected samples (Fig. 5B) and samples exposed to UV-inactivated EV71 (Fig. 5C) probably due to more total proteins loaded as seen from the total p38 and β-actin levels. However, it is evident in the trend of p38 MAPK activation profile that phospho-p38 MAPK levels were significantly increased upon EV71 infection (Fig. 5D). Hence, these results suggested that activation of p38 MAPK signalling pathway requires the active replication of EV71.

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