首页|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

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

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  • 国家科技期刊平台
  • NETL
  • NSTL
  • 万方数据
  • 维普
探索非洲猪瘟病毒(African swine fever virus,ASFV)调控细胞因子产生和细胞死亡的机制.利用NLRP3炎症小体表达系统,筛选发现ASFV-C84L蛋白诱导NLRP3炎症小体介导的IL-1β的分泌上调.首先,利用生物信息学分析C84L的结构信息,通过实时荧光定量PCR(qPCR)和间接免疫荧光试验(IFA)确定C84L蛋白在ASFV感染后的表达时序,细胞内定位;同时,用荧光素酶活性检测和蛋白免疫印迹(West-ern-blot)确定C84L蛋白参与调控p65的磷酸化以及IL-1β的成熟和细胞焦亡;然后,用碘化丙啶(PI)染色观察C84L蛋白诱导细胞的死亡情况;最后,使用qPCR和酶联免疫吸附试验检测C84L蛋白对IL-1β、IL-6、TNF-α等细胞因子转录和分泌的影响.结果显示:C84L为亲水性蛋白,与沙门菌效应蛋白SifA具有同源性;ASFV感染原代猪肺泡巨噬细胞4 h后,C84L mRNA表达水平逐渐上调,后续持续表达,第8小时达到顶峰;而且IFA结果显示,C84L蛋白在细胞质和细胞核中均有定位.荧光素酶和Western-blot结果显示,C84L促进p65的磷酸化并激活NF-κB启动子的活化.PI染色结果显示,C84L真核质粒转染细胞后诱导细胞死亡,Western-blot结果也显示C84L诱导Caspase-1成熟以及膜孔蛋白N-GSDMD(GSDMD剪切后的N端片段)剪切.将C84L真核质粒转染细胞后,IL-1β、IL-6、TNF-α等细胞因子转录水平呈剂量依赖性升高;与转录水平相似,C84L表达诱导IL-1β、IL-6、TNF-α分泌水平上调.结论:ASFV C84L蛋白通过激活NF-κB以及NLRP3炎症小体诱导IL-1β、IL-6、TNF-α等促炎细胞因子表达水平上调以及细胞焦亡的发生.
C84L protein of African swine fever virus up-regulates the expression of inflammatory factors by activating NLRP3 inflammasome
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.

African swine fever viruscytokinesC84LNF-κBNLRP3

唐静、马旭升、石正旺、代军飞、叶得河、王萌、马永华、郑海学

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甘肃农业大学 动物医学院,甘肃 兰州 730070

中国农业科学院 兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室,甘肃兰州 730000

非洲猪瘟病毒 细胞因子 C84L NF-κB NLRP3

国家重点研发计划项目兰州市科技计划项目兰州市科技计划项目甘肃农业大学基金项目甘肃农业大学基金项目甘肃农业大学基金项目甘肃农业大学基金项目甘肃省科技厅国际合作项目甘肃省自然科学基金项目国家生猪技术创新中心项目国家生猪技术创新中心项目"十四五"广东省"揭榜挂帅"项目中国农业科学院科技创新工程项目

2021YFD18013002022-2-1122017-4-103GAU-ZDKC-202216GAU-XKJS-2018-073GAU-KYQD-2017RCZX-11GAU-QDFC-2020-11144WCGA16918JR3RA167CARS-35NCTIP-XD/C032022SDZG02CAAS-ASTIP-2022-LVRI

2024

10.16656/j.issn.1673-4696.2024.0008

中国兽医科学
中国农业科学院兰州兽医研究所

中国兽医科学

CSTPCD北大核心
影响因子：0.524
ISSN：1673-4696
年,卷(期)：2024.54(1)
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