HnRNP K Interacts With Foot-and-mouth Disease Virus To Regulate Viral Replication And Its Molecular Mechanism

Foot-and-mouth disease?FMD?is a severe infectious disease of cloven-hooved animal species that is caused by foot-and-mouth disease virus?FMDV?.The disease severely compromises livestock production,ensuing high economic losses every year.FMDV belongs to the Aphthovirus genus of the Picornaviridae family.Similar to other picornaviruses,the FMDV genome lacks a 5'-cap structure,and its translation is controlled by an IRES located in the 5'UTR.Numerous studies have comfirmed that the regulation of IRES-mediated translation has been regarded as a critical step for picornaviruses infections,with important effects on virulence,pathogenicity,and tissue tropism.A better understanding of the regulation mechanisms of viral IRES-driven translation could therefore have important implications for elucidate the pathogenic mechanism of FMDV.Our previously research had isolated 8 IRES-associated cellular proteins,using a biotinylated FMDV IRES RNA pulldown assay followed by liquid chromatography-mass spectrometry/mass spectrometry?LC-MS/MS?analysis.In this study,we further investigated the role of heterogeneous nuclear ribonucleoprotein K?hnRNP K?.hnRNP K is a multifunctional protein that belong to member of the hnRNP family.hnRNPK interacts with nucleic acids or proteins involved in regulates transcription,translation,pre-mRNA splicing,RNA stability,chromatin remodeling and signal transduction.To verify the interactions of cellular hnRNP K with viral IRES,the RNA-protein pulldown assay was performed in BHK-21 cells,and Western blot analysis showed that the hnRNP K interacted with FMDV IRES.Further investigation revealed that the interaction between the FMDV IRES and hnRNP K was not restricted to one species or cell type.Furthermore,the competition assay revealed that hnRNP K interacted directly with the FMDV IRES.The hnRNP K directly binds to the FMDV IRES was confirmed by RNA pulldown assay was performed in the experssed hnRNP K.In addition,using RIP assays,we found that hnRNP K associates with the FMDV genomic RNA during FMDV infection.And immunofluorescence assay showed that hnRNP K colocalized with FMDV RNA in the cytoplasm.These results demonstrated that hnRNP K specifically interacts with FMDV IRES both in vitro and in vivo.Further research found that hnRNP K interacts with Domain II,III and IV of FMDV IRES through KH2 and KH3 domains.In the BHK-21 cells,the overexpression of hnRNP K decreased the expression of FMDV VP2protein,viral titers and viral RNA synthesis.In contrast,the knockdown of hnRNP K increased the expression of FMDV VP2 protein,viral titers and viral RNA synthesis.These results indicate that hnRNP K inhibits viral replication in the early stages of FMDV infection,but hnRNP K loses its inhibitory effect on viral replication in the later stages of FMDV infection.In addition,the overexpression of hnRNP K decreased the expression of EMCV VP1 protein,viral titers and viral RNA synthesis.These results suggest that hnRNP K may exert a broad-spectrum inhibitory effect on the type II IRES virus.Further investigation of the inhibitory mechanism of hnRNP K on FMDV replication showed that hnRNP K acts as a negative regulator of FMDV translation and replication by competing with the positive ITAF PTB for FMDV IRES binding.Conversely,FMDV has developed a strategy in which the viral 3C protease cleaves hnRNP K at the Glu-364 residue in a protease activity-dependent manner to antagonize the restriction of hnRNP K.Interestingly,the cleavage of hnRNP K yields two cleavage products with opposite functions,of which,the N-terminal cleavage product hnRNP K_1-364 retains a partial inhibitory effect on the IRES activity and FMDV replication,and the C-terminal cleavage product hnRNP K_364-465 becomes a positive regulator of FMDV replication.In conclusion,we identified an ITAF,hnRNP K,which negatively regulates FMDV replication by inhibiting viral IRES-mediated translation.Further investigation revealed that hnRNP K negatively regulates IRES-mediated translation by interfering with the recognition of the another positive ITAF,polypyrimidine tract binding protein?PTB?.Here,we also report that hnRNP K-mediated inhibition is antagonized by the viral 3C protease through cleavage of hnRNP K during FMDV infection.This study shed light on a molecular mechanisms that the cellular hnRNP K protein regulates viral IRES-driven translation and FMDV modify hnRNP K by using viral protease,and provides new insights into translational control during picornaviruses infection.