The Molecular Mechanism Of TRIM56 On Inhibition Of Porcine Epidemic Diarrhea Virus Replication

Porcine epidemic diarrhea virus(PEDV)belongs to the coronavirus family,a member of the genus?coronavirus,and is one of the main pathogens of porcine viral diarrhea.PEDV infection can cause Porcine epidemic diarrhea(PED)in pigs,especially for lactating piglets,which has a very high morbidity and fatality rate.It mainly causes severe diarrhea,vomiting,decreased appetite,dehydration and even death in piglets.Therefore,in-depth study of the host protein's mechanism against PEDV infection is of great significance for the development of antiviral drugs.The laboratory used proteomics sequencing technology to screen out multiple differentially expressed proteins after PEDV infected Marc-145 cells.Among them,it was found that the expression of TRIM56 gene,one of the TRIM family,was up-regulated.This study determined that TRIM56 has an effect on PEDV proliferation through overexpression and siRNA interference,and then focused on the molecular mechanism of TRIM56inhibiting PEDV replication,and obtained the following results:1.TRIM56 inhibits PEDV replication.In this study,PEDV was used to Marc-145cells were infected with PEDV.TRIM56 mRNA and protein levels in the cells were significantly up-regulated by RT-q PCR and Western blot analysis;the monkey TRIM56overexpression plasmid was constructed,and the overexpression of TRIM56 was detected after transfection into Marc-145 cells.the results show that overexpression of TRIM56 can significantly inhibit PEDV replication.PEDV replication level was significantly increased in TRIM56 knockdown Marc-145 cells.2.TRIM56 significantly up-regulates the expression of IFN-?and interferon-stimulated genes(ISGs).TRIM56 protein was overexpressed in Marc-145 cells,and the expression of type I interferon and cytokine were detected by RT-q PCR after PEDV infection.The results showed that overexpression of TRIM56 can significantly promote the expression of IFN-?and IL-8.Knockdown of TRIM56 protein only weakened the expression of IL-8,but did not affect the expression of IFN-?.These results indicate that overexpression of TRIM56 in Marc-145 cells can significantly induce IFN-?expression to inhibit PEDV replication;knockdown of TRIM56 has no effect on IFN-?expression,indicating that TRIM56 may also inhibit PEDV replication through other antiviral mechanisms.3.TRIM56 activates IRF3 and NF-?B signaling pathways.the expression of IRF3,I?B?and NF-?B phosphorylation were detected by Western blot in TRIM56overexpression or knockdown cells.The results showed that TRIM56 overexpression can significantly upregulate IRF3,I?B?and NF-?B phosphorylation levels,while TRIM56 konckdown has no effect on the expression of IRF3 phosphorylation,only reduces the levels of I?B?and NF-?B phosphorylation.These results indicate that overexpression of TRIM56 can induce the expression of IFN-?by regulating the IRF3and NF-?B signaling pathways.4.TRIM56 participates in the regulation of TLR3 innate immune signaling pathway.the expression of TLR3,TRIF and TRAF3 proteins were detected by Western blot infected in PEDV infected Marc-145 cells.The results showed that PEDV can activate the TLR3 signaling pathway;TRIM56 can up-regulate the protein expression level of TRAF3 and present an upward trend with the increase of the transfection dose;on the contrary,Knockdown of TRIM56 reduces the protein expression level of TRAF3.IFN-?,ISGs and cytokines were significantly up-regulated with dose dependeny in TRIM56 transfected cells.The above results indicate that TRIM56 is involved in the regulation of PEDV-induced TLR3 innate immune signaling pathway to inhibit PEDV replication.5.The N-and C-terminal domains of TRIM56 are necessary to inhibit PEDV replication.TRIM56?RING,?N,?C truncated mutant plasmid were constructed,NF-?B promoter activity were detected by dual luciferase,and IFN-?transcription level were detected by RT-q PCR,PEDV replication were detected by western blot,RT-q PCR and TCID₅₀ methods in TRIM56 or truncated mutant transfected Marc-145 cells.The results show that only the TRIM56 overexpression plasmid has the ability to activate NF-?B,IFN-?and inhibit the virus,indicating that the N and C-terminal domains of TRIM56 are necessary for inhibiting PEDV replication.In summary,TRIM56 protein is significantly up-regulated by PEDV infection.TRIM56 promotes the expression of TRAF3 protein by regulating the TLR3 signaling pathway,enhances the IRF3 and NF-?B signaling pathways,and finally induces the expression of IFN-?and ISGs.Innate immunity is the basic defense mechanism of the host against pathogen invasion.The experimental results show that TRIM56 protein can exert antiviral effects by regulating the innate immune signal pathway.