Effects Of Porcine Epidemic Diarrhea Virus 3C-Like Protease (3CL^pro) On MAVS-Mediated Apoptosis

Porcine epidemic diarrhea（PED）is a gastrointestinal infectious disease of pigs caused by PED virus（PEDV）.PEDV is classified into the Coronaviridae.The 3C-like protease(3CL^pro)is the main protease encoded by its Nsp5 gene,which plays an important role in virus proliferation and pathogenesis.In the course of antiviral infection,host cells inhibit the generation of virus through cell apoptosis,which plays a key role in the process of host antiviral infection.At present,it has been proved that a variety of coronavirus can induce apoptosis,and previous studies have shown that cell apoptosis caused by SARS-Co V is related to its 3CL^pro.However,the molecular mechanism of PEDV and its 3CL^proinducing cell apoptosis is still unclear.And MAVS is a critical factor in viral infection and innate immunity.Whether PEDV and its 3CL^proinduce cell apoptosis by regulating MAVS has not been reported yet.This study aims to analyze the molecular basis of PEDV and 3CL^proregulation of MAVS-mediated apoptosis,preliminarily clarify the role of 3CL^proin virus-host interaction,and lay a theoretical foundation for revealing the pathogenic mechanism of PEDV.1 PEDV epidemic strain JS2013 induced apoptosis of Vero cellsIn order to reveal whether PEDV epidemic strain JS2013 induces cell apoptosis and its related molecular mechanisms.Firstly,PEDV JS2013 was successfully amplified by Vero cells with 5μg/m L trypsin.Then we explored the mechanism of apoptosis induced by JS2013 in Vero cells by indirect immunofluorescence,flow cytometry,Hoechest stainning,CCK-8 and Western blot.The results showed that a small part of the virus successfully infected Vero cells at 6h after infection,typical syncytial lesions were observed at 24h after infection,and obvious apoptotic features were observed under transmission electron microscopy.Flow cytometry results showed that PEDV JS2013 induced Vero cell apoptosis in a time-dependent and dose-dependent manner,and significantly reduced cell activity.Western blot analysis showed that caspase pathway was activated after infection.And the proportion of apoptotic cells caused by PEDV epidemic strain JS2013 was significantly higher than that of vaccine strain CV777.These results indicate that PEDV epidemic strain JS2013 can induce Vero cell apoptosis,which is helpful to understand the pathogenic mechanism of PEDV and provide some reference for the prevention and control of PED.2 PEDV 3CL^proinduced cells apoptosisTo further reveal whether PEDV 3CL^procan induce apoptosis,we firstly constructed eukaryotic expression vector of 3CL^proof PEDV.Specific primers were designed according to the sequence of PEDV 3CL^proin Gen Bank.Then the gene was amplified by RT-PCR,and transfected into HEK 293T cells.The expression of this protein was identified by Western blot.And bioinformatics software was used to analyze the sequence,structure and function of 3CL^pro.The results showed that 3CL^prowas rich in glycosylation and phosphorylation sites.Then we further explored the mechanism of apoptosis induced by3CL^proin HEK 293T cells by Hoechest stainning,electron microscopy,flow cytometry,CCK-8 and mitochondrial membrane potential detection.The data showed that PEDV3CL^procan induce apoptosis,decrease cell viability and reduce mitochondrial membrane potential in HEK 293T cells.Taken together,our observation strongly suggested PEDV3CL^prohas the ability to induce apoptosis,which is provide data support for the study of the biological functions of PEDV 3CL^pro,and promote the research of small molecule anti-apoptotic drugs.3 The impact on inducing apoptosis by protease activity site mutations of PEDV3CL^proBased on the above findings,we further analyzed the effect of PEDV 3CL^proprotease activity on apoptosis.Firstly,the amino acid sequences of 3CL^proof several coronaviruses were compared,and two corresponding protease activity catalytic sites of PEDV 3CL^prowere His at position 41 and Cys at position 144 respectively.Two mutants,p CMV-H41A and p CMV-C144A were constructed based on the p CMV-3CL^proplasmid.After transfection,CCK-8,flow cytometry and mitochondrial membrane potential were used to investigate the effect of PEDV 3CL^proprotease activity on apoptosis.The results showed that the rate of apoptosis,the degree of inhibition of cell activity and the proportion of depolarization cells caused by the two mutants were significantly lower than that of 3CL^pro.These results indicated that PEDV 3CL^proinduced apoptosis was dependent on its protease activity.And the study provides data support for further study on the biological functions of PEDV 3CL^pro.4 Effect of PEDV 3CL^proon MAVS-mediated apoptosisIn order to further elucidate the effect of PEDV 3CL^proon MAVS-mediated apoptosis,we overexpressed the MAVS plasmid,the results showed that over expression of MAVS can induce cell apoptosis in a dose-dependent manner.3CL^proand its mutants plasmids were co-expressed with MAVS in HEK 293T cells to examine the rate of apoptosis,the data showed that 3CL^prosynergically promoted MAVS-mediated apoptosis and depended on its protease activity.Then we successfully obtained MAVS deletion cell lines by CRISPR/Cas9 gene editing technology.After transfection with 3CL^pro,the apoptotic cells were significantly reduced compared with the wild cells,indicating that MAVS were involved in the signaling pathway of cell apoptosis induced by 3CL^pro.The results of co-immunoprecipitation test showed that 3CL^promay indirectly affect the MAVS-mediated apoptosis pathway through the interaction with other proteins in the pathway.In this chapter,the effect of 3CL^proon MAVS-mediated apoptosis was preliminarily explored,which is helpful to provide data support for the study on the biological function of PEDV3CL^proin virus-host interaction.Through the above studies,we preliminarily explored the role of PEDV 3CL^proin apoptosis and its effect on MAVS-mediated apoptosis.This result contributes to a deeper understanding of the pathogenesis of PEDV and the interaction between the virus and host cells,and provides a new idea for the development of effective antiviral drugs and vaccines.