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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

Proposed mechanism of action of MINK in the EV71 replication cycle.EV71 infection stimulates MINK activation which in turn triggers the phosphorylation of p38 MAPK downstream. The phosphorylation of p38 MAPK triggers a kinase cascade which results in the cytoplasmic relocalisation of hnRNP A1. hnRNP A1 binds to the viral IRES and promotes the recruitment of ribosomes at the IRES at the 5’ untranslated region (UTR) of EV71 genome, stimulating the IRES-mediated viral protein translation.
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ppat.1004686.g009: Proposed mechanism of action of MINK in the EV71 replication cycle.EV71 infection stimulates MINK activation which in turn triggers the phosphorylation of p38 MAPK downstream. The phosphorylation of p38 MAPK triggers a kinase cascade which results in the cytoplasmic relocalisation of hnRNP A1. hnRNP A1 binds to the viral IRES and promotes the recruitment of ribosomes at the IRES at the 5’ untranslated region (UTR) of EV71 genome, stimulating the IRES-mediated viral protein translation.

Mentions: Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) classes have been identified as trans-acting factors that control translation initiation of various cellular and viral mRNAs at the IRES [60]. Among the hnRNP family, hnRNP A1 has been reported to modulate the IRES-mediated viral protein translation of various viruses such as the human rhinovirus (HRV) [61] and EV71 [20]. Although, hnRNP A1 localises predominantly in the nucleus, it is able to shuttle between the nucleus and cytoplasm in a regulated manner [62]. Infection of cells with HRV and EV71 has shown to result in the cytoplasmic relocalisation of hnRNP A1 where it interacts directly with the viral IRES sequences [20]. Apart from picornaviruses, cytoplasmic accumulation of hnRNP A1 has also been reported to play a role in the positive regulation of human immunodeficiency virus (HIV) [63] and Sindbis virus (SINV) [20] viral RNA translation. In uninfected cells, activation of the p38 MAPK pathway upon osmotic shock or UV irradiation has been revealed to result in a phosphorylation-dependent cytoplasmic accumulation of hnRNP A1 [35]. Furthermore, a separate study has also demonstrated that the p38 MAPK interacts and regulates the subcellular localisation of hnRNP A1 in a Mnk1-dependent manner in senescent cells [37]. The cytoplasmic relocalisation of hnRNP A1 after EV71 infection may therefore also be dependent on the p38 MAPK pathway and its downstream substrate Mnk1 as in uninfected cells. In our study, subcellular localisation studies unravelled the relationship between MINK protein expression and hnRNP A1 localisation in the cells. Interestingly, we have found that the silencing of MINK upon EV71 infection did not result in the cytoplasmic accummulation of hnRNP A1 which was usually observed in infected cells. The nuclear retention of hnRNP A1 could be due to either lower levels of EV71 replication as a result of MINK silencing or a block in nuclear export signal [64] brought about by the siRNA knockdown of MINK. Similarly, the inhibition of p38 MAPK with a specific p38 MAPK inhibitor (SB203580) also resulted in the accumulation of hnRNP A1 signals in the nucleus. Although we have demonstrated that inhibition of the MINK/p38 MAPK signalling pathway reduced the hnRNP A1 signals in the cytoplasm that was observed in control EV71-infected cells, we have no direct evidence suggesting that MINK plays a direct role on the cytoplasmic relocalisation of hnRNP A1 where it binds directly to the IRES sequences of the viral genome to promote the IRES-mediated translation of the EV71 viral RNA. In addition, we have also shown that despite its activation during EV71, p38 MAPK substrate Mnk1 was not involved in the regulation of EV71 protein synthesis and the cytoplasmic relocalisation of hnRNP A1. Hence, the exact mechanism of how MINK/p38 MAPK signalling pathway affected the cytoplasmic relocalisation of hnRNP A1 during EV71 infection and the p38 MAPK substrates involved needs to be further established. Nonetheless, we have shown in this study that a novel host kinase (MINK) mediates the cap-independent translation of EV71 RNA, possibly by modulating the subcellular localisation of hnRNP A1, which further supports its propagation (Fig. 9). As such, MINK can be further explored as potential antiviral target for the inhibition of EV71 viral replication at the viral protein translation 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)

Proposed mechanism of action of MINK in the EV71 replication cycle.EV71 infection stimulates MINK activation which in turn triggers the phosphorylation of p38 MAPK downstream. The phosphorylation of p38 MAPK triggers a kinase cascade which results in the cytoplasmic relocalisation of hnRNP A1. hnRNP A1 binds to the viral IRES and promotes the recruitment of ribosomes at the IRES at the 5’ untranslated region (UTR) of EV71 genome, stimulating the IRES-mediated viral protein translation.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004686.g009: Proposed mechanism of action of MINK in the EV71 replication cycle.EV71 infection stimulates MINK activation which in turn triggers the phosphorylation of p38 MAPK downstream. The phosphorylation of p38 MAPK triggers a kinase cascade which results in the cytoplasmic relocalisation of hnRNP A1. hnRNP A1 binds to the viral IRES and promotes the recruitment of ribosomes at the IRES at the 5’ untranslated region (UTR) of EV71 genome, stimulating the IRES-mediated viral protein translation.
Mentions: Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) classes have been identified as trans-acting factors that control translation initiation of various cellular and viral mRNAs at the IRES [60]. Among the hnRNP family, hnRNP A1 has been reported to modulate the IRES-mediated viral protein translation of various viruses such as the human rhinovirus (HRV) [61] and EV71 [20]. Although, hnRNP A1 localises predominantly in the nucleus, it is able to shuttle between the nucleus and cytoplasm in a regulated manner [62]. Infection of cells with HRV and EV71 has shown to result in the cytoplasmic relocalisation of hnRNP A1 where it interacts directly with the viral IRES sequences [20]. Apart from picornaviruses, cytoplasmic accumulation of hnRNP A1 has also been reported to play a role in the positive regulation of human immunodeficiency virus (HIV) [63] and Sindbis virus (SINV) [20] viral RNA translation. In uninfected cells, activation of the p38 MAPK pathway upon osmotic shock or UV irradiation has been revealed to result in a phosphorylation-dependent cytoplasmic accumulation of hnRNP A1 [35]. Furthermore, a separate study has also demonstrated that the p38 MAPK interacts and regulates the subcellular localisation of hnRNP A1 in a Mnk1-dependent manner in senescent cells [37]. The cytoplasmic relocalisation of hnRNP A1 after EV71 infection may therefore also be dependent on the p38 MAPK pathway and its downstream substrate Mnk1 as in uninfected cells. In our study, subcellular localisation studies unravelled the relationship between MINK protein expression and hnRNP A1 localisation in the cells. Interestingly, we have found that the silencing of MINK upon EV71 infection did not result in the cytoplasmic accummulation of hnRNP A1 which was usually observed in infected cells. The nuclear retention of hnRNP A1 could be due to either lower levels of EV71 replication as a result of MINK silencing or a block in nuclear export signal [64] brought about by the siRNA knockdown of MINK. Similarly, the inhibition of p38 MAPK with a specific p38 MAPK inhibitor (SB203580) also resulted in the accumulation of hnRNP A1 signals in the nucleus. Although we have demonstrated that inhibition of the MINK/p38 MAPK signalling pathway reduced the hnRNP A1 signals in the cytoplasm that was observed in control EV71-infected cells, we have no direct evidence suggesting that MINK plays a direct role on the cytoplasmic relocalisation of hnRNP A1 where it binds directly to the IRES sequences of the viral genome to promote the IRES-mediated translation of the EV71 viral RNA. In addition, we have also shown that despite its activation during EV71, p38 MAPK substrate Mnk1 was not involved in the regulation of EV71 protein synthesis and the cytoplasmic relocalisation of hnRNP A1. Hence, the exact mechanism of how MINK/p38 MAPK signalling pathway affected the cytoplasmic relocalisation of hnRNP A1 during EV71 infection and the p38 MAPK substrates involved needs to be further established. Nonetheless, we have shown in this study that a novel host kinase (MINK) mediates the cap-independent translation of EV71 RNA, possibly by modulating the subcellular localisation of hnRNP A1, which further supports its propagation (Fig. 9). As such, MINK can be further explored as potential antiviral target for the inhibition of EV71 viral replication at the viral protein translation 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