GSK-3 Mediated ?-catenin Regulating The Proliferation Of PEDV

Porcine epidemic diarrhea virus(PEDV)belongs to the type I coronavirus,which is a single stranded plus strand RNA(ssRNA)virus.PED is susceptible to all ages of pigs,and the mortality in7-day-old piglets is 100%.In recent years,the outbreak of variant PEDV has caused a serious impact on the pig industry.Viruses can cause a variety of signaling pathways changes in cells after infection and the PI3K/AKT/GSK-3 signaling pathway plays an important role in regulating virus infection.?-catenin is one of the downstream substrate of GSK-3,which can be degraded by phosphorylation of GSK-3 and plays an important role in the process of viral transcriptional regulation.The results of preliminary experimentes in our laboratory showed that the activation of PI3K/AKT/GSK-3 signaling pathway could inhibit the proliferation of PEDV in Vero cells.In order to further explore the molecular mechanism of the PEDV replication,we used inhibitors to inhibit protein expression and overexpress eukaryotic expression vectors to explore the effects of PI3K/AKT/GSK-3 signaling pathway and downstream signal factors on PEDV replication.The results of this experiment are as follows:1.After Marc-145 cells infected with PEDV,the phosphorylation level of AKT at Ser473 increased and the level of GSK-3?/?(Ser21/9)phosphorylation protein increased.Mean while,the content of ?-catenin increased.After inhibiting PI3K/AKT pathway with specific inhibitor LY294002,phosphorylation of AKT(Ser473)was inhibited,GSK-3?/?(Ser21/9)phosphorylation was decreased,and ?-catenin content was also reduced(indicating GSK-3 activity Increased).PEDV's proliferation on Marc-145 cells was inhibited.The results showed that the activation of PI3K/AKT signaling pathway could promote the proliferation of PEDV in Marc-145 cells.2.After inhibiting the GSK-3 activity with inhibitor CHIR-99021 on the Marc-145 cells,the expression of ?-catenin increased,and the replication of the virus was inhibited.The results of this study showed that the content of ?-catenin was increased after GSK-3 activity reduced,and it could inhibit the proliferation of PEDV.N protein is the nucleocapsid protein of virus,which has an effect on viral replication,transcription,and translation,and plays an important regulatory role in the process of virus proliferation.In order to further clarify the molecular mechanism of GSK-3 regulating PEDV proliferation,we over-expressed PEDV N protein by transfected p3 x Flag-N eukaryotic expression plasmid after inhibiting GSK-3 activity.The results showed the expression of N protein decreased after adding inhibitor CHIR-99021 on 293 T cells and Marc-145 cells,while the expression of N protein increased on Vero cells.3.After inhibiting the Wnt/?-catenin pathway with inhibitor Salinomycin on Marc-145 cells,the expression of ?-catenin decreased significantly,while the replication of PEDV increased.The results showed that the increase of ?-catenin inhibited the replication and proliferation of PEDV.In order to further explore whether the replication of the PEDV N protein was affected by ?-catenin,wecotransfected eukaryotic expression plasmid p3×Flag-?-catenin and p3×Flag-N into 293 T cells.The results showed that ?-catenin has a significant inhibitory effect on the expression of N protein.In summary,on the one hand,the proliferation of PEDV can be promoted by activating PI3K/AKT signaling pathway.On the other hand,the activated PI3K/AKT signaling pathway can increase the expression of ?-catenin by inhibiting the activity of GSK-3,and further inhibits the proliferation of PEDV by inhibiting the expression of N protein.