USP21增强EV-A71 2A^(pro)稳定性负向调控RLR信号通路促进EV-A71复制  

USP21 negative regulates RLR pathway by stabilizing EV-A712A^(pro) to promote EV-A71 replication

作　　者：杨歆煜 唐梦园 车治萍 陈艳 彭阳[1] 马锦洪[1] 史伟峰[1] 周围 Yang Xinyu;Tang Mengyuan;Che Zhiping;Chen Yan;Peng Yang;Ma Jinhong;Shi Weifeng;Zhou Wei(Department of Clinical Laboratory,the Third Affiliated Hospital of Soochow University,Changzhou 213003,China;Department of Clinical Laboratory,Changzhou Children′s Hospital,Changzhou 213018,China;Department of Military Sperm Bank,Changhai Hospital,Shanghai 200433,China)

机构地区：[1]苏州大学附属第三医院(常州市第一人民医院)医学检验科,常州213003 [2]常州市儿童医院检验科,常州213018 [3]上海长海医院军人精子库,上海200433

出　　处：《中华实验和临床病毒学杂志》2025年第1期18-26,共9页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金青年科学基金项目(82102473);常州市科技支撑计划(CJ20245005,CJ20245014);常州市"十四五"卫生健康高层次人才培养工程—拔尖人才(2024CZBJ014)。

摘　　要：目的探究泛素特异性蛋白酶21(ubiquitin-specific protease 21,USP21)在肠道病毒A组71型(enterovirus group A type 71,EV-A71)感染中的作用。方法采集EV-A71感染患儿与健康儿童各24例外周全血,分离出外周血单个核细胞(peripheral blood mononuclear cell,PBMC),实时荧光定量PCR(real-time fluorescence quantitative PCR,qPCR)检测PBMC中USP21 mRNA表达水平。采用qPCR或蛋白印迹试验(western blot,WB)观察过表达USP21或敲除USP21后对EV-A71复制的影响;WB检测EV-A71结构蛋白VP1、RIG-I样受体(RIG-I-like receptor,RLR)信号通路关键分子蛋白水平及干扰素调节因子3(interferon regulatory factor 3,IRF3)磷酸化。免疫共沉淀(co-immunoprecipitation,Co-IP)分析USP21对EV-A71非结构蛋白2A蛋白酶(2A^(pro))泛素水平的影响。结果与健康儿童相比,EV-A71感染患儿PBMC中USP21 mRNA表达明显升高。过表达USP21后显著提升EV-A71所致的细胞病变效应,上调VP1 mRNA和蛋白水平并促进EV-A71复制,细胞活性也随USP21的转染水平增高而明显下降;而敲除USP21基因后发现EV-A71 VP1 mRNA水平较对照组显著下调。另外,过表达USP21并不影响EV-A712A^(pro)的转录水平,但可以上调2A^(pro)的蛋白表达,同时降低2A^(pro)的泛素化水平,抑制线粒体抗病毒信号蛋白(mitochondria antiviral signaling protein,MAVS)、黑色素瘤分化相关基因5(melanoma differentiation-associated gene 5,MDA5)的蛋白表达以及IRF3的磷酸化水平,下调干扰素-β(interferon-β,IFN-β)mRNA水平。敲除USP21可挽救EV-A71感染导致的MAVS和MDA5蛋白水平下调,促进IRF3的磷酸化并上调IFN-βmRNA水平。结论USP21可通过降低2A^(pro)的泛素化水平,降低MAVS和MDA5蛋白表达,负向调控干扰素信号通路而促进EV-A71复制。ObjectiveTo investigate the role of ubiquitin-specific protease 21(USP21)in enterovirus group A type 71(EV-A71)infection.MethodsPeripheral blood mononuclear cells(PBMC)were obtained from a cohort of 24 children infected with EV-A71 and 24 healthy children.Expression of USP21 was determined by real-time fluorescence quantitative PCR(qPCR).Additionally,the impact of USP21 overexpression or knockout on EV-A71 replication was evaluated using a combination of qPCR and western blot(WB)analysis.Furthermore,WB was employed to measure the levels of EV-A71 structural protein VP1,phosphorylated interferon regulatory factor 3(IRF3)and other key molecules in the RIG-I-like receptor(RLR)signaling pathway.Co-immunoprecipitation(Co-IP)was utilized to investigate the effects of USP21 on the ubiquitin levels of EV-A71 nonstructural protein 2A^(pro)tease(2A^(pro)).ResultsIn comparison to healthy children,the expression of USP21 mRNA in PBMC of children infected with EV-A71 was notably elevated.The overexpression of USP21 significantly enhanced the cytopathic effects induced by EV-A71,upregulated levels of VP1 mRNA and protein,and facilitated EV-A71 replication,leading to a decrease in cell activity with increasing levels of USP21 transfection.Following the knockout of the USP21 gene,the VP1 mRNA levels were significantly declined in comparison to the control group.Furthermore,the overexpression of USP21 was found to have no impact on the transcriptional activity of EV-A712A^(pro).However,it was observed to enhance the expression of 2A^(pro) protein,reduce the ubiquitination of 2A^(pro),suppress the protein levels of mitochondrial antiviral signaling protein(MAVS)and melanoma differentiation-associated gene 5(MDA5),as well as decrease the phosphorylation of IRF3.Additionally,the induction of IFN-βmRNA by EV-A71 infection was downregulated.ConclusionsUSP21 has been shown to enhance the replication of EV-A71 through the downregulation of 2A^(pro) ubiquitination,suppression of MAVS and MDA5 protein expression,and inhibition of the int

关 键 词：肠道病毒A组71型 泛素特异性蛋白酶21 2A蛋白酶 RLR信号通路 

分 类 号：R73[医药卫生—肿瘤]