非洲猪瘟病毒C84L蛋白通过激活NLRP3炎症小体上调炎症因子的表达OACSTPCD

This study explored the mechanism by which African swine fever virus(ASFV)up-regulates the production of pro-inflammatory cytokines and cell death.Using NLRP3 inflammasome expression sys-tem,we found that ASFV-C84L protein could induce the NLRP3 inflammasomes-mediated pro-inflammatory cytokines upregulation.Firstly,the structure of C84L was analyzed by bioinformatics,and the expres-sion stage and intracellular localization of C84L protein after ASFV infection were determined by real-time fluorescent quantitative PCR(qPCR)and indirect immunofluorescence assay(IFA).Meanwhile,luciferase activity assay and Western-blot confirmed that C84L protein was involved in p65 phosphory-lation,IL 1β maturation,and pyroptosis.Then the cell death was observed by propidium iodide(PI)staining during C84L expression.Finally,qPCR and enzyme-linked immunosorbent assay were used to de-tect the effect of C84L protein on the transcription and secretion of IL-1β,IL-6 and TNF-α etc.The re-sults showed that C84L was a hydrophilic protein with homology to Salmonella effector protein SifA,and the expression of C84L mRNA in primary porcine alveolar macrophages was gradually increased at 4 h after ASFV infection,followed by continuous expression and reached the peak at 8 h.In addition,IFA results showed that C84L protein was localized in both cytoplasm and nucleus.The results of luciferase and Western-blot showed that C84L activated p65 phosphorylation and the NF-κB promoter.PI staining showed that C84L expression induced cell death,and Western-blot results also showed that C84L induced the maturation of Caspase-1 and N-GSDMD(the N-terminal cleaved fragment of GSDMD).After C84L eukaryotic plasmid transfected cells,the transcription levels of IL-1β,IL-6,TNF-α and other cytokines increased in a dose-dependent manner.Similarly,C84L eukaryotic plasmid transfected cells could increase IL-1β,IL-6 and TNF-α secretion levels.In conclusion,ASFV C84L protein induces the upregulation of IL-1β,IL-6,TNF-α and other cytokines as well as pyroptosis by activating NF-κB and NLRP3 inflammasome.