SARS-CoV-2 ORF7a通过靶向IKKβ激活NF-κB信号通路促进细胞炎症因子释放  

SARS-CoV-2 ORF7a promotes release of inflammatory cytokines by targeting IKKβto activate NF-κB signaling pathway

作　　者：牟露敏 龙启舟 邓东青 程金芝[1] 聂映 吴家红 MOU Lumin;LONG Qizhou;DENG Dongqing;CHENG Jinzhi;NIE Ying;WU Jiahong(The key Laboratory of Environmental Pollution Monitoring and Disease Control School of Public Health Guizhou Medical University,Guiyang 550025,China;The First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang 550002,China;Department of Medical Parasitology College of Basic Medicine Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学公共卫生与健康学院环境污染监测与疾病控制重点实验室,贵阳550025 [2]贵州中医药大学第一附属医院,贵阳550002 [3]贵州医科大学基础医学院现代病原生物学特色重点实验室,贵阳550025

出　　处：《中国免疫学杂志》2024年第4期714-719,共6页Chinese Journal of Immunology

基　　金：贵州省科技计划项目(黔科合基础[2019]1021号);贵州省教育厅青年科技人才成长项目(黔教合KY字[2018]213)。

摘　　要：目的:探究SARS-CoV-2辅助蛋白ORF7a介导NF-κB激活,进而诱导炎症因子产生的分子机制。方法:双荧光素酶报告基因试验检测ORF7a对NF-κB激活的影响,qRT-PCR检测ORF7a对细胞中炎症因子表达的影响,Western blot、核质分离及免疫荧光技术检测ORF7a蛋白对p65磷酸化及入核的影响,免疫共沉淀及免疫荧光技术检测ORF7a的作用靶标蛋白。结果:报告基因试验表明ORF7a显著激活NF-κB启动子活性,并呈剂量依赖性(P<0.001),而对AP-1报告基因的激活无明显影响。ORF7a显著上调细胞因子TNF-α、IL-β及IL-8 mRNA表达(P<0.05)。Western blot结果显示ORF7a显著增强p65蛋白磷酸化(P<0.05)及p65的入核(P<0.01)。免疫共沉淀实验发现ORF7a与NF-κB信号通路分子IKKβ蛋白存在相互作用,免疫荧光实验也证实ORF7a与IKKβ具有共定位。结论:SARS-CoV-2 ORF7a通过靶向IKKβ激活NF-κB信号通路,进而促进细胞中炎症因子的释放。Objective:To investigate the molecular mechanisms by which SARS-CoV-2 accessory protein ORF7a mediating NF-κB activation and thus inducing inflammatory cytokines production.Methods:Effects of ORF7a on NF-κB promoter activity was analyzed by luciferase reporter assay.qRT-PCR was used to analyze expressions of inflammatory cytokines.Effects of ORF7a on phosphorylation and nuclear-translocation of p65 were determined by Western blot and immunofluorescence.Co-immunoprecipitation and immunofluorescence assay were performed to investigate potential target s of ORF7a.Results:Luciferase reporter assay showed that ORF7a activated NF-κB promoter activity in a dose-dependent manner(P<0.001),while has no effect on activation of AP-1 reporter gene.ORF7a significantly upregulates expressions of TNF-α,IL-1βand IL-8 mRNA levels(P<0.05).Western blot showed that ORF7a markedly increased phosphorylation of p65 protein(P<0.05)and p65 nuclear localization(P<0.01).The interaction between ORF7a and IKKβprotein of NF-κB signaling pathway was found by immunoprecipitation assay,and the co-localization of ORF7a and IKKβwas also confirmed by immunofluorescence assay.Conclusion:SARS-CoV-2 ORF7a targets IKKβto promote active NF-κB pathway to the release of inflammatory cytokines.

关 键 词：SARS-CoV-2 ORF7a NF-ΚB信号通路 炎症因子 

分 类 号：R392.1[医药卫生—免疫学]