Study On Molecular Mechanism Of Porcine Deltacoronavirus Regulating Unfolded Protein Response

Porcine deltacoronavirus(PDCo V)is a single-stranded positive-stranded RNA virus of Nidovirales and the Coronavirus family.PDCo V infection can cause diarrhea and death in nursing piglet.PDCo V,for the first time,broke out in the United States in 2014 and quickly spread to several states.Subsequently,the existence of PDCo V was discovered in many pig farms in Multiple countries,which caused certain economic losses to the pig industry and attracted great attention from the pig industry.In addition,some studies have reported that PDCo V can infect calves and chickens,and recent studies have confirmed that it can infect humans,suggesting its potential for cross-species infection.Currently,PDCo V is unique member of the deltacoronavirus genus that can be continuously passaged in vitro.Therefore,PDCo V possesses a great basic research value as a good model for studying the deltacoronavirus genus.Endoplasmic reticulum(ER)is the key sites of protein synthesis,processing,maturation and intracellular transport tasks.Virus used the endoplasmic reticulum membrane system of the host to replicate.The accumulation of a large number of viral proteins in the endoplasmic reticulum will exceed the normal regulatory capacity of the endoplasmic reticulum,and then induce endoplasmic reticulum stress.Host cell will initiate an effective response,the unfolded protein response(UPR).Early studies have shown that many viruses can finely regulate UPR to achieve the optimal replication and pathogenic goals.As a newly emerged porcine enteric coronavirus,the relationship between PDCo V and UPR is unclear.Therefore,this study uses the PDCo V CHN-HN-2014 strain as a representative to explore the relationship between PDCo V infection and UPR.The specific research content is as follows:1.PDCo V infection activates the IRE1/XBP1 pathwayLLC-porcine kidney cells(LLC-PK1)and porcine small intestinal mucosal epithelial cells(IPI-2I)were inoculated with PDCo V,followed by the detection of spliced XBP1 at different time points by RT-PCR.The results showed that as the virus infection progressed,the amount of XBP1(s)produced by cleavage gradually increased.Quantitative real-time PCR experiment results showed that the m RNA level of XBP1(s)gradually increased.The above results indicate that PDCo V infection can activate the IRE1/XBP1 pathway.In addition,the m RNA levels of downstream effector molecules EDEM1 and ERDJ4 were detected by Quantitative real-time PCR experiments,and the results showed that PDCo V infection did not upregulate the expression of EDEM1 and ERDJ4 suggesting that the host cells used certain mechanism to antagonize the expression of these effector molecules.2.PDCo V infection activates the PERK pathwayLLC-PK1 and IPI-2I were inoculated with PDCo V,and the expression level of phosphorylated e IF2? protein at different time points after PDCo V infection was detected by Western blot experiment.The results showed that as the virus infection progressed,the expression of phosphorylated e IF2? protein gradually increased.At the same time,the results of Quantitative real-time PCR experiments showed that the m RNA expression levels of ATF4,GADD34 and CHOP were significantly upregulated as the infection time prolonged.Combined to the method of puromycin-labeled nascent protein,western blot results showed that PDCo V infection significantly inhibited host protein translation in a dose-dependent manner.These results indicate that PDCo V infection activates the PERK pathway and inhibits host protein translation.3.PDCo V infection does not activate the ATF6 pathwayLLC-PK1 and IPI-2I were inoculated with PDCo V,and the m RNA levels of ATF6pathway-related effector molecules GRP78,GRP94,Calnexin,Calreticulin and ERp57 at different time points after PDCo V infection were detected by Quantitative real-time PCR experiments and none of the relevant effector molecules were activated.Western blot results showed that the protein levels of ATF6 pathway associated effector molecules,including GRP78,GRP94,Calreticulin,PDI,and transcription factor p50ATF6,did not significantly increase after PDCo V infection.4.The effects of the IRE1 and PERK pathways on virus replicationTo further determine whether the IRE1 and PERK pathways affect the replication of PDCo V,through drug treatment,overexpression experiments and RNA interference experiments,the results showed that inhibiting the IRE1 pathway did not affect the proliferation of PDCo V,while interfering with the PERK pathway significantly inhibited the proliferation of PDCo V.To further explore the role of PERK pathway in PDCo V proliferation,e IF2? was overexpressed on LLC-PK1 cells.The results showed that e IF2?expression promotes the upregulation of viral m RNA levels.These results indicate that PERK pathway is beneficial to virus replication.5.PDCo V NS6 protein activates 3 pathways of UPRIn addition,to further determined which proteins encoded by PDCo V are involved in the regulation of UPR,all the proteins encoded by PDCo V are screened through the UPRE/ERSE dual luciferase reporter system.The results showed that NS6 protein has the most significant ability to upregulate the activity of UPRE and ERSE promoters among all the proteins.Subsequently,we manly focus on the molecular mechanism of NS6 protein on regulation of UPR.The result show that overexpression of NS6 can activate 3 pathways of UPR in HEK-293 T cells.