The Mechanisms Of Middle East Respiratory Syndrome Coronavirus Non-structural Protein Nsp14 Inhibits Lung Cell Apoptosis

Research backgroundMiddle East Respiratory Syndrome Coronavirus(MERS-CoV)is the sixth known coronavirus that infects humans.Because of its extremely high fatality rate and the occurrence of small-scale infections,it is a serious threat to human health.With the pandemic of COVID-19 around the world,the coronavirus has aroused the interest of many researchers.However,we have not yet fully understood the pathogenic mechanism of the seven known coronaviruses that can infect humans.MERS-CoV is a 30.1K virus with a single-stranded positive-stranded RNA as its genetic material.Its genome consists of 11 open reading fragments,which encode the replicase polyprotein pplab,structural protein and accessory protein.The replicase polyprotein pplab is cleaved by protease into 16 nonstructural proteins(nsp)nspl-nsp16.Studies have shown that nonstructural proteins play an important role in virus replication and antagonize the host's natural immune response.Apoptosis is a well-studied method of cell death,and it plays an important role in the process of virus infection.When the virus invades the host,apoptosis of the infected cells will limit the replication of the virus.Inhibiting host cell apoptosis in the early stage of infection can increase the ability of the virus to replicate.Many articles have reported relevant research on coronavirus protein and cell apoptosis.It is believed that MERS-CoV can cause lung or kidney cell apoptosis which may be an important cause of organ failure in patients.Therefore,screening and exploring the effects of non-structural proteins in MERS-CoV on cell apoptosis is not only conducive to our understanding of its pathogenicity,but also provides theoretical and experimental basis for the selection of antiviral drug targets.Research purposes1.Screen MERS-CoV nsp that can regulate lung cell apoptosis;2.Explore the mechanism of MERS-CoV nsp affecting cell apoptosis and its effect on virus replication;3.Provide new target for the prevention and treatment of MERS-CoV.Research methods1.Carry out lentivirus packaging in 293T cells by the calcium phosphate method,collect the virus fluid,infect A549 cells,and observe the infection efficiency under a fluorescence microscope after 48 hours;2.Inducing cell apoptosis by culturing in serum-free medium,using JC-1 and Mito-Tracker mitochondrial membrane potential dyes to confirm the establishment of serum-free culture-induced lung cell apoptosis model by flow cytometry,fluorescence microscopy and Western Blot;3.Under the conditions of complete medium and serum-free medium for 12h and 24h,flow cytometry was used to detect the effect of nsp 14 on A549 cell apoptosis,and Western Blot was used to detect the activation of AKT/mTOR,P65,Caspase-3 and Caspase-8,Flow cytometry to detect A549 cell cycle;4.Use endogenous apoptosis inducer ABT-737 to treat 293T cells,detect the effect of nsp 14 on 293T cell apoptosis by flow cytometry,and detect the changes of apoptosis molecules by quantitative PCR and Western Blot;5.Quantitative PCR to detect the effects of nsp 14 on apoptosis-related molecules BAX,SMAD7,APAF-1,BCL-2,BCL2L2 and HSP701 A,and Western Blot to detect the effects of nsp 14 on apoptosis-related proteins BAX,BCL2L1,BCL2L2,and BCL-2;6.Under the model of culturing cells in serum-free medium,use the mitochondrial membrane potential indicator Mito-Tracker to detect the effect of nsp 14 on the mitochondrial membrane potential of A549 cells by flow cytometry and confocal microscopy,and use the Seahorse cell energy metabolism analyzer Detect the effect of nsp14 on the mitochondrial function of A549 cells;7.Use the PCDH vector to construct the p53-HA overexpression plasmid,and detect the interaction between p53 and nsp14 in 293T cells;8.Construct BAX overexpression plasmid on PCDH vector,BAX was transducted to nsp14-infected A549.Mito-Tracker was used to detect the mitochondrial membrane potential;9.Use pcDNA3.1 vector to construct nsp14-GFP and nsp14-His overexpression plasmids;10.Transfect nsp14-His plasmid and organelle marker-fluorescence fusion plasmid into 293T cells,observe the subcellular localization and co-localization with multiple organelle by using confocal microscope;11.The morphological changes of 293T cells caused by VSV infection were photographed by electron microscopy.CCK8 was used to detect the effect of nsp14 on the proliferation of 293T cells after VSV infection,quantitative PCR to detect the effect of nsp14 on virus replication after VSV infection,and flow cytometry to detect VSV infection of 293T cells.The effect of nsp14 on mitochondrial membrane potential after 12h.Research results1.Construction and verification of nsp vectorWe constructed the MERS-CoV nsp expression plasmid using the lentiviral vector PCDH.Observation by fluorescence microscope showed that the cell transfection efficiency was close to 100%.The lentiviral disease was collected,and the collected lentiviral solution was used to infect A549 cells for 48 hours and then photographed.The results showed that the infection efficiency of A549 cells was high.2.Serum-free medium-induced cell apoptosisWe use serum-free culture medium to induce lung cell apoptosis as a model.After JC-1 staining,fluorescence microscope and flow cytometry were used to detect apoptosis of H460 cells and decrease of mitochondrial membrane potential.The mitochondrial membrane potential indicator Mito-Tracker staining,flow cytometry results showed that serum-free induced a decrease in the mitochondrial membrane potential of A549 cells.At the same time,Western Blot results showed that serum-free medium induced the activation of exogenous cell apoptosis molecule Caspase8.3.nsp14 inhibits the activation of Caspase3After culturing the cells in a serum-free medium for 12 hours,we screened that nsp14 can inhibit the early and late apoptosis of A549 cells,but it has no significant effect on the A549 cell cycle.Western Blot results showed that nsp14 had no significant effect on NF-?B and AKT/mTOR signaling pathway.nsp14 can inhibit the expression of Cleaved-Caspase3 induced by serum-free cell culture medium,but has no obvious effect on the expression of Pro-Caspase3,Pro-Caspase8 and Cleaved-Caspase8.4.nsp14 inhibits endogenous cell apoptosis induced by ABT-737The results of flow cytometry showed that nsp14 inhibited ABT-737-induced endogenous cell apoptosis.The results of quantitative PCR and Western Blot showed that nsp14 inhibited the expression of BAX in 293T cells.5.nsp14 regulates apoptosis related moleculesQuantitative PCR results showed that,compared with the control group,nsp14 inhibited the mRNA expression of pro-apoptotic molecules BAX,SMAD7 and APAF-1,and promoted the inhibition of the mRNA expression of apoptosis molecules BCL-2,BCL2L2 and HSP701A.Western Blot results showed that nsp14 significantly inhibited the expression of BAX,promoted the expression of BCL-2,and had no significant effect on BCL-W,BCL-xl and other molecules.6.nsp14 inhibits apoptosis through maintaining mitochondria potentialWe used a mitochondrial membrane potential indicator to stain A549 cells cultured in serum-free medium to observe the changes in membrane potential.The results of flow cytometry and focusing microscopy showed that the mitochondrial membrane potential of the nsp14 group was higher than that of the control group.Since the decrease of cell mitochondrial membrane potential can damage mitochondrial function,we tested the pressure of cell mitochondria.Seahorse results showed that when A549 cells were cultured in serum-free medium,the mitochondrial respiration capacity,ATP production capacity,maximum respiration value and spare respiration capacity of the nsp14 group were significantly higher than those of the control group7.Interaction between nsp14 and p53We constructed a p53-HA overexpression plasmid using the PCDH vector and overexpressed exogenous p53-HA and nsp14 in 293T cells.The IP results indicated that nsp14 interacted with p53.8.Over-expression of BAX ameliorated nsp14-induced the suppression of A549 cell apoptosisWe constructed a BAX overexpression plasmid using the PCDH vector and infected A549 cells stably expressing nsp 14 with BAX lentivirus.Flow cytometry was used to detect changes in mitochondrial membrane potential.The results showed that overexpression of BAX eliminated the ability of nsp14 to maintain the mitochondrial membrane potential.9.Construction of overexpression vector of nsp14-GFP and nsp14-His fusion plasmidIn order to further explore the mechanism of nsp14 inhibiting cell apoptosis,we used pcDNA3.1 vector to construct nsp14-GFP and nsp14-His fusion plasmids.10.Subcellular localization of nsp14After transfecting the fluorescent plasmids of nsp14-His and organelle Marker into 293T cells,the results of confocal microscopy showed that nsp14 was distributed in the cytoplasm in the form of dots,not in the nucleus.There is no co-localization in the late endosomes mitochondria,Golgi and lysosomes.11.nsp14 promotes VSV virus replication by maintaining cell viabilityAfter we infected 293T cells expressing nsp14 with VSV that can induce endogenous cell apoptosis,the CCK8 results showed that after VSV infection,the cell viability of the nsp14 group was higher than the control group at 12h,and the cell viability of the nsp14 group was lower than the control group at 24h.The results of quantitative PCR showed that the viral genome expression in the nsp14 group was higher than that in the control group at 12h and 24h after VSV infection.The results of flow cytometry showed that when VSV infected 293T cells for 12 hours,the mitochondrial membrane potential of the nsp14 group was significantly higher than that of the control group.Conclusion and significance1.This study found that the MERS-CoV non-structural protein nsp14 can inhibit lung cell apoptosis,suggesting that nsp14 is beneficial to virus replication by maintaining host cell viability in the early stage of virus infection;2.nsp14 inhibits endogenous cell apoptosis by maintaining mitochondrial membrane potential;3.nsp14 is a potential target for drug candidates to prevent and treat MERS-CoV.