The Mechanism Of ID1 Suppressing Foot-and-mouth Disease Virus Serotype O Replication

Foot and Mouth Disease(FMD)is an acute,febrile,highly contact infectious disease which is caused by foot and mouth disease virus(FMDV).The host of FMDV is mainly pigs,cattle,deer,goats,sheep and other hoofed animals.The disease has many transmission routes and spreads rapidly.It has been outbreaks and epidemics for many times in the world,causing huge economic losses.Foot-and-mouth disease virus belongs to the genus Aphthovirus of the family Picornaviridae.Its genome is a single positive-stranded RNA with a length of about 8.5 kb.Seven serotypes have been identified of FMDV,and there are no cross-protection among the serotypes.Inhibitor of DNA binding 1(ID1)is a transcriptional regulator,which is mainly involved in cell differentiation,cell cycle progression,epithelial-mesenchymal cell transformation,angiogenesis,cell stem maintenance,chemoresistance,tumorigenesis and metastasis and some other biological processes.Up to now,there are few reports on the regulation mechanism of ID1 on viral replication.In order to reveal the molecular regulatory mechanism of the hosts against viral infection,LC-MS/MS was used to detect the protein expression profiles in host cells at different time points after FMDV challenged.Western blot was used to verify the significant differentially expressed proteins,and ID1 protein was selected for further study.Firstly,the ID1 expression levels at different time points post FMDV infection in different cells was determined,and the results showed that the expression of ID1 protein was firstly up-regulated and then down-regulated.When ID1 gene was knocked out in the cell lines using CRISPR-Cas9 system,compared with the control cell lines,FMDV proliferation was significantly promoted in ID1 gene knockout cells,which indicating that knockout of ID1 increased FMDV replication.Secondly,in order to verify whether these results are consistent with the in vivo experiments results,we constructed ID1 knockout transgenic mice.Compared with the control groups,ID1 knockout mice had a significantly reduced survival rate,a significantly shortened survival time,and the pathological sections of the heart showed that the lesions were much more serious.Thirdly,in order to explore the mechanism that FMDV downregulates ID1 expression,cells were treated with MG132,an inhibitor of the ubiquitin-proteasome system,and the results revealed that MG132 treatment significantly inhibited the downregulation of ID1 induced by FMDV infection,and the downregulation of ID1 was mediated by a E3 ligase Cdh1,which could bind and degrade ID1 protein.Finally,in order to make a further explanation of the molecular mechanism of ID1 in inhibiting the replication of foot-and-mouth disease virus,we found that ID1 could regulate the activity of transcription factor FOXO1 through co-immunoprecipitation assays.The results of luciferase reporter assays and ChIP experiments demonstrated that FOXO1 directly acted on IRF3 to regulate the expression of interferon,and eventually regulate foot-and-mouth disease virus proliferation.In addition,ssc-miR-381-3p,ssc-miR-4331-5p,ssc-miR-7137-5p and ssc-miR-4334-5p could bind to the 3'-UTR of ID1 mRNA through predictive analysis of bioinformatics softwares.Together,these results revealed the molecular mechanism of ID1 regulating FMDV replication,laid the foundation for elucidating the infection mechanism and immune escape strategies of FMDV,and provided the theoretical basis for virus immune prevention and antiviral drug development..