Molecular Mechanism Of TRIM8 Targeting Regulation Of Porcine Epidemic Diarrhea Virus N Protein Ubiquitination To Inhibit Viral Replication

Porcine epidemic diarrhea(PED)is a highly contagious intestinal infectious disease in pigs caused by the porcine epidemic diarrhea virus(PEDV),which is characterized by vomiting,watery diarrhea,and dehydration,and has a high mortality rate of 80%to 100%in affected piglets.There is no specific treatment for PED,and the emergence of new strains has led to an increasing failure of prevention and control,further exacerbating the difficulty and cost of control,which seriously threatens the healthy development of China’s pig industry.In the long run,through genetic improving the resistance of pigs to PEDV by means of methods will produce permanent effects.Screening and identification of PEDV resistance genes and related molecular markers is an important prerequisite for carrying out porcine epidemic diarrhea resistance breeding.Tripartite motif-containing proteins(TRIMs)play an important regulatory role in the process of virus replication in host cells,and can directly or indirectly regulate virus replication.We previously performed transcriptome sequencing on the jejunum tissue of PEDV-infected piglets,and found that the expression level of TRIM8 gene,a member of the TRIMs family,was significantly up-regulated,speculating that it may be an important regulatory gene for the replication of PEDV infection.To investigate the function and mechanism of TRIM8 in the process of PEDV infection,this study used intestinal porcine epithelial cell line(IPEC-J2)as a research model.Firstly,the expression changes of TRIM8 gene at different time points of PEDV infection were detected to reveal the relationship between TRIM8 expression and PEDV infection.Then,TRIM8 overexpression cells were constructed to further explore the regulatory effect of TRIM8 overexpression on PEDV infection.Next,transcriptome sequencing technology was used to investigate the effect of TRIM8 overexpression on the expression of PEDV infection-related genes and signaling pathways.Immunoprecipitation technology was employed to detect the interaction between TRIM8 protein and PEDV N protein,and the molecular mechanism of TRIM8 mediating the degradation of PEDV N protein in host cells was revealed through ubiquitination experiments.The main results of this study are as follows:1.TRIM8 inhibits the replication of PEDV in host cells.By detecting the expression of TRIM8 gene at different time points of PEDV infection using qRT-PCR and Western blot,the results showed that the expression level of TRIM8 gene was significantly up-regulated(P<0.05)after 12 h,24 h,and 48 h of virus infection,with the highest expression level at 24 h(P<0.01).Western blot experiments further verified the expression level of TRIM8 at different infection time points,which was consistent with the trend of its mRNA expression.To explore the function of TRIM8,this experiment constructed TRIM8 gene overexpression IPEC-J2 cells.PEDV infected TRIM8 overexpression cells and control cells,and the virus infection was detected by qRT-PCR,TCID50 and Western blot experiments.The results showed that the PEDV M gene copy number and virus titer in TRIM8 overexpression cells were significantly lower than those in the control group(P<0.01),and the expression level of PEDV N protein was also significantly reduced.2.Analysis of TRIM8 overexpression-related genes and signaling pathways in PEDVinfected cells.Transcriptome sequencing was performed on the PEDV-infected group,the PEDV-infected TRIM8 overexpression group,and the virus-uninfected control cells.The principal component analysis of the sequencing data showed that the three experimental groups were significantly divided into three clusters.A total of 2153 differentially expressed genes were screened by the differential expression analysis between the PEDV infection group and the control group,including 1195 up-regulated genes and 958 down-regulated genes;a total of 153 genes were screened between the PEDV-infected TRIM8 overexpression group and the PEDV-infected group differentially expressed genes,including 126 up-regulated genes and 27 downregulated genes.Finally,taking the intersection of the differentially expressed genes screened by the above two groups,a total of 22 genes with opposite expression trends were screened,among which members of the chemokine family and zinc finger protein family were screened,for example,the expression of CXCL2 gene in epithelial cells play an important role in cell-mediated tissue inflammation;ZNF527 and ZNF658 genes may be involved in inhibiting virus replication in host cells.Functional annotation and enrichment analysis were performed on the differentially expressed genes screened between the PEDV-infected group and the PEDV-infected TRIM8 overexpression group.It was found that the significantly enriched Go terms mainly included defense response and immune response;through signal pathway analysis,it was found that,the significantly enriched KEGG mainly included RIG-Ⅰ-like receptor signaling pathway and NOD-like receptor signaling pathway.Combined with biological functions,8 genes including HSPA6,ISG15,IFI6,and MX1 were selected from the differentially expressed genes for quantitative verification.The expression trend of qRT-PCR results was consistent with that of RNAseq results.3.TRIM8 targets PEDV N protein ubiquitination and degradation.Using Co-IP interaction technology to reveal the interaction between porcine TRIM8 protein and PEDV N protein,Western blot results showed that the abundance of PEDV N protein expression showed a dose-dependent decrease in TRIM8;using cycloheximide(CHX)to inhibit protein synthesis during the process,it was found that when the PEDV N protein synthesis pathway was inhibited,the degradation rate of PEDV N protein was significantly accelerated,revealing that TRIM8 can promote the degradation process of PEDV N protein;HEK293T cells were treated with proteasome inhibitor MG 132 and lysosome inhibitor Leupeptin,respectively,The expression level of PEDV N protein was significantly increased after MG132 treatment,but the addition of Leupeptin had no significant effect,indicating that TRIM8 can mediate the degradation of PEDV N through the proteasome system rather than lysosome.In order to explore whether TRIM8,as an E3 ubiquitin ligase,mediates PEDV N protein ubiquitination,this experiment constructed wild-type Ub,mutant K48 and K63 vectors,and co-transfected with TRIM8 expression vectors and PEDV N protein tag vectors,the results showed the ubiquitination levels of PEDV N protein in the wild-type Ub and mutant K48 groups were consistent,indicating that TRIM8 degrades PEDV N protein through K48 polyubiquitination.In summary,this study found that TRIM8 can inhibit the replication of PEDV in host cells,and the overexpression of TRIM8 activates signaling pathways such as RIGⅠ-like receptors;in addition,TRIM8 targets and regulates PEDV N protein,and the expression abundance of N protein shows TRIM8 dose-dependently decreased,and the mechanism analysis showed that TRIM8 targeted PEDV N protein K48 ubiquitination site to mediate the degradation of N protein through the ubiquitin-proteasome system.This study revealed the regulatory function and mechanism of TRIM8 during PEDV infection,enhanced the understanding of the molecular mechanism of the interaction between PEDV and host cells,and provided TRIM8 as an important candidate gene related to PEDV resistance in porcine epidemic diarrhea resistance breeding a theoretical basis is provided.