Molecular Mechanism Of Heterogeneous Nuclear Ribonucleoprotein ?/L On Foot-and-mouth Disease Virus Replication

As a critical element in the 5'UTR of picornavirus,the internal ribosome entry site(IRES)binds to various cellular proteins to participate in viral replication processes.In the previous study,we found that the C351 mutation of the IRES domain 4 confers foot-and-mouth disease virus(FMDV)with an ideal temperature-sensitive attenuation(ts&att)phenotype,but the molecular mechanism of this remains unclear.In this study,we found that C351G mutation,which located in the stem-loop of IRES,is one of binding regions of polypyrimidine binding protein(PTB)to interact with IRES,and PTB is a critical host factor during IRES-mediated translation initiation.Therefore,we speculate that C351G mutation may affect the binding ability of IRES to PTB,leading to the ts&att phenotype of FMDV.Further,we found that C351G mutation impaired the interaction between IRES and PTB,and then caused IRES temperature dependent translation defects,which is the molecular basis of ts&att phenotype of FMDV.The cellular protein PTB,also known as heterogeneous nuclear ribonucleoprotein I(hnRNP I),is an important member of the hnRNP family.However,there is limited information about other host proteins participating in FMDV replication,except for PTB.Thus,to examine host proteins interacting with the FMDV IRES,cell proteins were pulldown by biotinylated FMDV IRES RNA,followed by MALDI-TOF MS analysis.Totally,8 abundant proteins,including hnRNP L,were identified to be associated specifically with the FMDV IRES in BHK-21 cells.The hnRNP L,a multifunctional RNA-binding protein implicated in mRNA regulation reported previously,was selected for further investigation here.Firstly,the specific interaction between hnRNP L and FMDV IRES was verified by competition assay and confocal observation.Furthermore,it was found that hnRNP L interacts with FMDV IRES domain 4-5 through its RRM3-4.This RNA-protein interaction significantly inhibited the replication of FMDV in BHK-21 cells.However,the inhibitory effect of hnRNP L on FMDV growth was based on its binding to IRES,but it was not related to IRES-mediated translation.Therefore,the binding of IRES only provides a spatial site for hnRNP L to interact with other host cells or viral factors to inhibit viral RNA synthesis.We further found that hnRNP L negatively regulates FMDV replication by inhibiting viral RNA synthesis.It is known that PTB can stabilize IRES structure and is necessary for FMDV replication.This suggests that hnRNP L may inhibit FMDV replication by interacting with PTB to affect the stability of PTB and lead to IRES degradation.However,the results show that hnRNP L does not affect the stability of IRES.Further studies have shown that hnRNP L interacts with viral polymerase 3Dpol in FMDV-infected cells,and this interaction is FMDV RNA-dependent,suggesting that hnRNP L may exist in RNA replication complex to play a negative role for FMDV,but its mechanism is still unclear.Considering that hnRNP L interacts with FMDV IRES through its RNA recognition motifs RRM3-4,and some studies have showed that hnRNP L RRM3-4 may bind to two separate binding sites within the same RNA by inducing RNA looping.Therefore,we speculate that hnRNP L may change its conformation by binding IRES and recruit other proteins or noncoding RNAs to affect viral RNA synthesis in replication complexes.In conclusion,we found that the C351G mutation of IRES causes a temperature-dependent translation defect by impairing its binding to PTB,resulting in the ts&att phenotypes of FMDV.In addition,we also found a cellular protein hnRNP L as a novel ITAF that interacts with FMDV IRES,and it negatively regulates FMDV replication by inhibiting viral RNA synthesis.This study of hnRNP I and hnRNP L will help us to further understand the pathogenesis of FMDV,the molecular mechanism of virulence and host tropism of FMDV.