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.
Highlights
Human enterovirus 71 (EV71), a member of the Picornaviridae family and genus Enterovirus, is the major causative agent of hand-foot-and-mouth disease (HFMD)
We show that a serine/threonine kinase, Misshapen Nck-interacting kinase (NIK)-related kinase (MINK), plays a role in the replication of Enterovirus 71 (EV71) by stimulating the p38 mitogen activated protein kinase (p38-Mitogen-activated Protein Kinase (MAPK)) pathway which in turns promotes the translation efficiency of EV71 viral protein synthesis
As the synthesis of viral proteins is crucial for the replication of the virus during infection, discovery of a crucial host kinase in this process may provide insights on the replication of EV71
Summary
Human enterovirus 71 (EV71), a member of the Picornaviridae family and genus Enterovirus, is the major causative agent of hand-foot-and-mouth disease (HFMD). The genomic RNA is translated into more viral polyproteins in a cap-independent manner and the polyproteins are subsequently processed into the structural capsid proteins and non-structural proteins [11]. The 5’ UTR of the EV71 genomic RNA contains a cloverleaf structure involved in viral RNA replication and an internal ribosomal entry site (IRES) which directs viral protein translation in a cap-independent manner [12,13]. A number of ITAFs have been identified to interact with picornavirus IRES and mediate translation initiation of the viral polyprotein These ITAFs include polypyrimidine tract-binding protein (PTB) [15,16,17], heterogeneous nuclear ribonucleoprotein E (hnRNP E) [18], far-upstream element-binding protein 1 (FBP1) and FBP2 [19]. Among these ITAFs, hnRNP A1[20],and FBPs [19] have been reported to interact with EV71 IRES
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