The Role Of Karyopherin α6 Protein In The Replication Of PEDV And The Mechanisms Of Microbiota-derived Metabolites Against PEDV Replication

Porcine epidemic diarrhea virus（PEDV）is a member of the Alphacoronavirus,which is highly infectious and can cause vomiting,diarrhea,dehydration and even death in piglets,seriously affecting the healthy development of the pig industry in China.Although vaccination is the main tool to prevent and control PED,the low viral titer of PEDV and the constant variation of strains still make the prevention and control of PEDV very challenging.Therefore,it is urgent to study the infection mechanism and new prevention and control strategies of PEDV.In addition,due to the complexity of PED prevention and control,there is no effective treatment method in clinical practice,so it is of great importance to explore the drug target of PEDV for the prevention and control of PEDV.This study focuses on the role of karyopherinα6 in replication of PEDV and the mechanism of microbiota-derived metabolites against PEDV.The main contents of this study are as follows:1.The regulation of karyopherinα6 on PEDV replication.Karyopherinα（KPNA）can transport macromolecular proteins from the cytoplasm to the nucleus through the nuclear pore complex,allowing them to function in the nucleus.PEDV N protein has a role in regulating viral RNA synthesis,viral particle assembly and promoting viral replication.Previous studies have confirmed that PEDV N protein can localized to the nucleus,but the mechanism of its nuclear entry has unclear.Based on proteomics analysis,our group detected the protein level of KPNA6 after PEDV infection of IPEC-J2 cells by Western Blot and found that the protein level of KPNA6 increased after PEDV infection;we found a possible interaction between KPNA6 protein and N protein by NLStradamus online software prediction.After recombinant expression of KPNA6 protein and N protein in HEK293T cells,the co-localization of KPNA6 protein and N protein were confirmed by confocal laser scanning microscope.These results demonstrated that the expression of KPNA 6 protein promoted the nuclear entry of N protein during PEDV infection of IPEC-J2 cells.By overexpression of KPNA6 protein in IPEC-J2 cells and infection with PEDV,KPNA6 promoted the replication of PEDV,and by RNA interference of KPNA6 protein expression in IPEC-J2 cells and infection with PEDV,the results showed that interference of KPNA6 inhibited PEDV replication.Therefore,this study revealed that PEDV promoted nuclear entry of N protein with KPNA6 through its interaction and viral replication,elucidated the replication regulation mechanism of PEDV from a new perspective,and provided new strategy for anti-PEDV drug.2.Butyrate limits the replication of PEDV.Short-chain fatty acids（SCFAs）are gut microbiota-derived metabolites that regulate intestinal function through various mechanisms to enhance the intestinal barrier and immune function.In order to explore the effect of SCFAs on PEDV replication,IPEC-J2cells were pre-treated with acetate,propionic,and butyrate with PEDV infection.q PCR,Western Blot,and TCID₅₀were used for detection.The results show that butyrate displayed a better effect than other SCFAs on limiting PEDV replication in IPEC-J2 cells.The expression of interferon and interferon stimulating genes was detected by q PCR in cells treated with butyrate with PEDV infection.The results showed that butyrate induced the expression of type III interferon and OAS1 and ISG15.To explore the mechanism of butyrate promoting type III interferon production,GPR43 in IPEC-J2 cells was blocked by GPR43 inhibitor GLPG0974 and RNA interference.Butyrate with GLPG0974 or interference GPR43 were co-pretreat IPEC-J2 cells and infected PEDV respectively.The results showed that both GLPG0974 and interfering GPR43 promoted PEDV replication.To investigate the activation of NF-κB downstream of GPR43 receptor by butyrate,IPEC-J2 cells were treated with NF-κB inhibitor and infected with PEDV.The results showed that NF-κB inhibitor restored the inhibitory effect of butyrate on PEDV,and butyrate activated IκBαphosphorylation in IPEC-J2 cells.These results indicate that butyrate activates the NF-κB signaling pathway through GPR43 receptor to promote interferon production,thereby inhibiting PEDV replication.This study revealed that butyrate has the potential to be an antiviral component for inhibiting PEDV replication.3.LCA exerts anti-viral activities against PEDV.Bile acids（BAs）are synthesized in the liver and metabolized in the intestine by the gut microbiota.Studies have shown that BAs can regulate cellular immunity and affect the process of viral infection.To investigate the effect of BAs on PEDV replication,q PCR,Western Blot and TCID₅₀methods were used to detect the effects of four BAs-cholic acid（CA）,chenodeoxycholic acid（CDCA）,deoxycholic acid（DCA）and lithocholic acid（LCA）on PEDV infection in IPEC-J2 cells.The results showed that all four BAs inhibited PEDV replication,and LCA showed excellent anti-PEDV activity,and the inhibitory effect was concentration-dependent.In-depth study revealed that LCA mainly inhibited the replication stages of PEDV infection in IPEC-J2 cells,and we found that LCA could activate the transcription of type III interferon and interferon-stimulated genes OAS1,ISG15 and CXCL10.To explore the mechanism by which LCA promotes type III interferon production,we blocked the FXR and TGR5 receptors with the inhibitors DY268 and SBI-115,respectively.The results showed that blocking TGR5 was more effective in inhibiting type III interferon expression and restoring PEDV replication than blocking the FXR receptor.These results suggest that LCA inhibits PEDV replication by promoting interferon production through the TGR5 receptor.This study demonstrated that LCA can be used as a potential anti-PEDV strategy.Collectively,this study uncover that KPNA6 promotes the entry of N into the nucleus and replication of PEDV through its interaction with N protein,this research provides perspective on the replication regulation mechanism of PEDV.On the other hand,this study confirmed that microbiota-derived metabolites butyrate and LCA inhibit PEDV replication.In summary,these results reveal the mechanism for regulating PEDV replication and provide novel strategy for the design and development of antiviral drug targets.