The CREB And AP-1 Dependent CCN1 Regulates PEDVinduced Cell Apoptosis Inhibiting Virus Replication

Porcine epidemic diarrhea virus（PEDV）is a member of the family Coronaviridae and genus alphacoronavirus,which mainly causes Porcine epidemic diarrhea（PED）.PED is a common intestinal infectious disease in the pig industry,mainly causing severe diarrhea,dehydration,loss of appetite,weight loss and other characteristic symptoms in sick pigs.All ages are susceptible to this disease,which mainly caused high lethality and seriously hinders the sustainable development of the pig industry.However,the prevention and treatment of this disease has not been resolved.In recent years,the study of PEDV pathogenic mechanism is few.To clarify the pathogenic mechanism of PEDV and effectively prevent PEDV,the relationship between virus and host protein were in-depth exploration.In the early stage of the experiment,a number of differentially expressed proteins were screened from the high-throughput sequencing results of PEDV-infected Marc-145 cells.CCN1 is one of the differentially expressed proteins.As a multifunctional protein,CCN1 can regulate a variety of intracellular activity.In this study,we explore the expression of CCN1 after PEDV infection and its transcriptional mechanism,and the effects of CCN1 protein on PEDV proliferation and its specific mechanism,and obtained the following results:1.PEDV infection up-regulates the expression of host protein CCN1.Using RT-qPCR and Western blot found that the m RNA and protein levels of CCN1 were increased after PEDV infection in Marc-145 cells and Vero cells.The CCN1 promoter vector and truncation mutant vector were construct,the core region of the CCN1 promoter in response to PEDV stimulation was screened through dual-luciferase reporter gene method.The result found that the CCN1promoter-985～203 region fluorescence activity was significantly elevated after PEDV infection.The transcription factor binding sites were deleted in the CCN1 promoter region of-985～203 to construct deletion mutants,and dual-luciferase reporter gene detection showed that transcription factors AP-1 and CREB could increase the expression of CCN1 in response to PEDV.The expression of CCN1 decrease by add AP-1 and CREB inhibitors.The results is consistent with the above results.By adding signaling pathway inhibitors to block related pathways in cells,the upstream signals regulating AP-1 and CREB were screened.The results showed that the effect of PEDV-induced CCN1 expression decrease when p38 and PKA pathways were inhibited.PEDV upregulates the expression of CCN1 by activate p38 and PKA to regulate the transcription factors AP-1 and CREB.2.CCN1 protein regulates PEDV replication through apoptosis pathway.The CCN1recombinant plasmid was transfected into Marc-145 cells.RT-qPCR and western blot detection showed that overexpression of CCN1 inhibited the expression of PEDV-N m RNA and protein.The virus titer in the culture supernatant was detected by TCID₅₀.The titer of PEDV was attenuated when CCN1 overexpression,whereas PEDV-N m RNA and protein expression increased when CCN1 interference,and the titer was enhanced.Flow cytometry showed that the apoptosis rate increased after CCN1 overexpression,and the apoptosis rate decreased after CCN1 interference.Western blot analysis showed that overexpression of CCN1 promoted caspase 3 cleavage,whereas interference with CCN1 inhibited caspase 3cleavage.By detecting the expression of death receptor pathway-related proteins（Fas,Fas L,caspase 8）and mitochondrial pathway-related proteins（Bax,Bcl-2,caspase 9）,it was found that CCN1-induced apoptosis is through the mitochondrial pathway,rather than the death receptor pathway.After treatment with p53 pathway inhibitor（PFT-α）,the regulation of apoptosis by CCN1 regulates apoptosis was blocked,indicating that the regulation of apoptosis by CCN1 was p53-dependent.And it was found that the effect of CCN1 inhibiting PEDV-N protein was also reversed.Then,by adding an apoptosis inhibitor（Z-VAD-FMK）to detect PEDV-N,the results found that the inhibitory effect of CCN1 on PEDV-N was weakened,indicating that the regulation of CCN1 on PEDV is through the apoptotic pathway.In summary,PEDV infection activates the p38 and PKA to regulate the activation of transcription factors AP-1 and CREB,and induces the transcription and expression of the host protein CCN1,which regulates mitochondrial apoptosis through the p53 pathway and inhibits PEDV replication,providing a new basis for exploring the relationship between PEDV and host protein CCN1.