中国兽医科学2024，Vol.54Issue(1)：1-11,11.DOI:10.16656/j.issn.1673-4696.2024.0008
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
C84L protein of African swine fever virus up-regulates the expression of inflammatory factors by activating NLRP3 inflammasome
摘要
Abstract
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.关键词
非洲猪瘟病毒/细胞因子/C84L/NF-κB/NLRP3Key words
African swine fever virus/cytokines/C84L/NF-κB/NLRP3分类
农业科技引用本文复制引用
唐静,马旭升,石正旺,代军飞,叶得河,王萌,马永华,郑海学..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[J].中国兽医科学,2024,54(1):1-11,11.基金项目
国家重点研发计划项目(2021YFD1801300) （2021YFD1801300）
兰州市科技计划项目(2022-2-112,2017-4-103) （2022-2-112,2017-4-103）
甘肃农业大学基金项目(GAU-ZDKC-202216,GAU-XKJS-2018-073,GAU-KYQD-2017RCZX-11,GAU-QDFC-2020-11) （GAU-ZDKC-202216,GAU-XKJS-2018-073,GAU-KYQD-2017RCZX-11,GAU-QDFC-2020-11）
甘肃省科技厅国际合作项目(144WCGA169) （144WCGA169）
甘肃省自然科学基金项目(18JR3RA167) （18JR3RA167）
国家生猪技术创新中心项目(CARS-35,NCTIP-XD/C03) （CARS-35,NCTIP-XD/C03）
"十四五"广东省"揭榜挂帅"项目(2022SDZG02) （2022SDZG02）
中国农业科学院科技创新工程项目(CAAS-ASTIP-2022-LVRI) （CAAS-ASTIP-2022-LVRI）
