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Study On The Effects And Mechanism Of Integrin αVβ3 During Porcine Deltacoronavirus Infection

Posted on:2023-07-09Degree:DoctorType:Dissertation
Country:ChinaCandidate:X H JinFull Text:PDF
GTID:1520306809451574Subject:Veterinary science
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Porcine deltacoronaviruses(PDCoV),which belongs to the family of Coronaviridae and the genus of deltacoronavirus,is an enveloped virus with a single-stranded positive-sense RNA genome.PDCoV mainly causes diarrhea,vomiting,dehydration and even death of piglets,which leads to huge economic losses for swine industry.As a novel swine enteropathogenic coronavirus in 2014,current studies on PDCoV mainly focus on virus isolation,identification,epidemiological investigation,and the establishment of detection method.However,its entry pathway,cell receptors,and the pathogenesis are still unclear.Therefore,in this study,the entry pathway of PDCoV HNZK-02 virus isolated in our laboratory was studied,and the host receptors or cofactors of PDCoV were screened and verified.The role of ITGαVβ3 in PDCoV infection was systematically analyzed,and the molecular mechanism of ITGαVβ3 in PDCoV infection was demonstrated.The main research contents are as follows:1.PDCoV infects host cells through endocytosis and exogenous protease pathways.In order to study the invasion pathway of PDCoV infection in host cells,ST and LLC-PK cells were used as PDCoV-infected models.q RT-PCR,TCID50 and IFA assays were used to confirm that the endocytosis inhibitor Baf-A1 could inhibit the infection of PDCoV in a dose-dependent manner,suggesting that PDCoV could infect host cells through endocytosis.The isolation and proliferation of PDCoV require the involvement of exogenous protease,but the role of exogenous protease in virus replication is still unclear.Therefore,we studied the role of trypsin in PDCoV infection,and found that PDCoV could also infect host cells without trypsin,but the addition of trypsin could significantly improve the infectious efficiency of virus.Also,the trypsin could still promote virus infection even in the presence of Baf-A1 inhibitor,indicating that exogenous protease could mediate PDCoV entry from cell membrane surface to host cells.These results indicated that PDCoV infected host cells through endocytosis and membrane surface pathway mediated by exogenous protease.2.Screening and preliminary identification of PDCoV receptor candidate molecules on ST cell membrane.Receptor proteins on cell membrane are required during the entry of PDCoV,no matter through which pathway.However,the receptors of PDCoV on susceptible cells are still unknown.In this study,cell membrane proteins of ST cells were isolated,and the receptors of PDCoV were screened by VOPBA and LC-MS/MS assay.The obtained cell membrane proteins were analyzed according to the protein score and peptide matching degree,and then combined with the existing researches on other viral receptors,ITGαV,ITGα3,ITGβ1,ITGβ3,VIM,MYH9 and HSP70 were selected for further verification.First,the results of q RT-PCR assay showed that m RNA levels of the other genes except ITGα3 were up-regulated during PDCoV infection.Subsequently,it was confirmed by si RNA assay that si VIM and si MYH9 did not affect PDCoV infection,while si HSP70 reduced the replication of PDCoV.The ITGαVβ1 and ITGαVβ3 inhibitors could inhibit PDCoV replication,but ITGαVβ3 was more potent.HSP70,ITGαVβ1 and ITGαVβ3 were identified as the cell receptors of PDCoV infection.3.ITGαVβ3 plays an important role in the adhesion,entry and release of PDCoV.In order to investigate the role of ITGαVβ3 in the process of PDCoV infection,Western Blot assay was used,and the results showed that the protein expression level of ITGαVβ3 was up-regulated after PDCoV infection.Images obtained by confocal laser microscopy showed that PDCoV co-located with ITGαVβ3 protein on the cell surface.Moreover,ITGαVβ3 inhibitor could inhibit PDCoV infection in a dose-dependent manner,and we further confirmed that ITGαVβ3 contributed to promote virus replication by si RNA and CRISPR-Cas.In order to further study the role of ITGαVβ3 in PDCoV infection,ITGαVβ3 inhibitor was added at different stages of PDCoV infection.It was found that ITGαVβ3 was involved in the virus adsorption,entry and release stages,and ITGαVβ3 was involved in the virus entry and exit process.4.ITGαVβ3 promotes PDCoV infection by activating the FAK-PI3K-AKT signaling pathway.The FAK-PI3K-AKT signaling pathway can be activated by ITGαVβ3 to promote virus replication during virus infection.To determine whether ITGαVβ3 could activate the FAK-PI3K-AKT signaling pathway and figure out its molecular mechanism during PDCoV infection,q RT-PCR and Western Blot were used.The results showed that PDCoV could activate FAK,PI3 K and AKT in a dose-dependent manner.The expression of AKT was inhibited after PI3 K inhibitor treatment,indicating that AKT was the downstream molecule of PI3 K.Further studies showed that PI3 K,AKT and ITGαVβ3 inhibitors could inhibit the replication of PDCoV.And the ITGαVβ3 inhibitor inhibited the phosphorylation of FAK and AKT.These results suggest that the infection of PDCoV requires the activation of ITGαVβ3-FAK-PI3K-AKT signaling pathway.5.PDCoV induces inflammatory response by activating ITGαVβ3-FAKPI3K-AKT-NF-κB signaling pathway.Previous studies in our laboratory found that PDCoV infection could induce the host to produce high levels of inflammatory cytokines both in vitro and in vivo.In order to study the molecular mechanism of PDCoV and inflammatory response,RNA-seq was used to analyze the differential gene expression at 12 h after PDCoV infection.The results showed that most of the differentially expressed genes were antiviral genes and inflammation-related genes,and KEGG analysis showed that the NF-κB signaling pathway were activated after PDCoV infection.Western Blot assay confirmed that PDCoV infection promoted the phosphorylation and nucleation of p65 protein by activating IκBα,and then up-regulated the expression levels of inflammatory factors(IL-8,IL-10 and TNF-α).Moreover,the inhibitor of NF-κB inhibited the expression of inflammatory factors,suggesting that PDCoV infection induced the expression of inflammatory factors through the activation of NF-κB signaling pathway.Studies showed that integrin further activates downstream NF-κB through the FAK-PI3K-AKT signaling pathway after receiving foreign signal stimulation,and then mediating the occurrence of inflammatory response.Therefore,we speculated that inflammatory factors induced by PDCoV infection were related to ITGαVβ3-FAK-PI3K-AKT and NF-κB signaling pathway.q RT-PCR and Western Blot assay confirmed that the ITGαVβ3,PI3 K and AKT inhibitors could inhibited the expression of inflammatory factors and NF-κB,as well as the phosphorylation and nucleation of p65,respectively.These results suggest that PDCoV could up-regulate the expression of inflammatory factors by activating ITGαVβ3-FAK-PI3K-AKT-NF-κB signaling pathway.In conclusion,PDCoV infects host cells through endocytosis and exogenous protease-mediated pathway.ITGαVβ3 plays a role in the adsorption,entry and release stages of PDCoV.ITGαVβ3 promotes the PDCoV infection by activating the FAK-PI3K-AKT signaling pathway,and PDCoV induces inflammatory response by activating ITGαVβ3-FAK-PI3K-AKT-NF-κB signaling pathway.The results of this study provide an important theoretical basis for further understanding of the interaction between PDCoV and host,further research on the immune pathogenesis,the development of anti-PDCoV drugs and effective vaccines of PDCoV.
Keywords/Search Tags:Porcine Deltacoronavirus (PDCoV), ITGαVβ3, FAK-PI3K-AKT signaling pathway, NF-κB signaling pathway, Inflammatory cytokines
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