牛病毒性腹泻病毒Npro蛋白与宿主IRF7相互作用机制的研究  

Mechanism of IRF7 regulation by bovine viral diarrhea virus Npro protein

作　　者：曹唱唱 项安琪 陈珊 王雨欣 王静 王爽 陈斐 宋厚辉 姜胜 宋泉江 CAO Chang-chang;XIANG An-qi;CHEN Shan;WANG Yu-xin;WANG Jing;WANG Shuang;CHEN Fei;SONG Hou-hui;JIANG Sheng;SONG Quan-jiang(Zhejiang Agriculture and Forestry University,College of Animal Science and Technology-School of Animal Medicine,Zhejiang Key Laboratory of Applied Technology Research on Green Ecological Healthy Breeding of Livestock and Poultry,Zhejiang Engineering Research Center of Animal Health Internet Detection Technology,Zhejiang International Science and Technology Cooperation Base of Animal Medicine and Health Management,and China-Australia Joint Laboratory of Animal Health Big Data Analysis,Hangzhou 311300,China;Xinzhuang Animal Husbandry and Veterinary Station,Zhaoyuan City,Yantai 265400,China)

机构地区：[1]浙江农林大学动物科技学院/动物医学院/浙江省畜禽绿色生态健康养殖应用技术研究重点实验室/动物健康互联网检测技术浙江省工程研究中心/浙江省动物医学与健康管理国际科技合作基地/中澳动物健康大数据分析联合实验室,浙江杭州311300 [2]招远市辛庄畜牧兽医站,山东烟台265400

出　　处：《中国预防兽医学报》2024年第12期1222-1229,1283,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金青年科学基金项目(31802258);浙江省自然科学基金探索一般项目(LY22C180003);浙江农林大学科研发展基金(2018FR015);浙江省新苗人才计划(2022R412A025)。

摘　　要：本团队前期研究发现干扰素调节因子7(IRF7)参与牛病毒性腹泻病毒(BVDV)在宿主细胞中的复制。为进一步研究BVDV与IRF7之间的相互作用机制,本实验经PCR扩增BVDV N^(pro)和IRF7基因后,分别构建真核表达质粒pCMV-Flag-IRF7和pCMV-HA-BVDV^(Npro),并分别经PCR和测序鉴定均正确。将pCMV-Flag-IRF7转染牛睾丸(BT)细胞后感染BVDV,24 h后分别采用western blot和RT-qPCR检测BVDV E2、IRF7蛋白的表达和BVDV 5'UTR、IRF7mRNA转录水平。结果显示,与BT细胞相比,过表达IRF7的BT细胞在感染后BVDV E2蛋白的表达水平极显著下调(P<0.001),且BVDV 5'UTR基因的转录水平极显著降低(P<0.001);与未感染BVDV的过表达IRF7组相比,感染BVDV后IRF7蛋白表达水平极显著下调(P<0.001),但IRF7 mRNA的转录水平无明显差异。进一步以MOI 0.1的BVDV感染IRF7过表达的BT细胞24 h后,采用RT-qPCR检测IRF7、IFN-α和IFN-βmRNA的转录水平。结果显示,与感染BVDV的BT细胞组相比,BVDV感染过表达IRF7的BT细胞组IRF7 mRNA的转录水平极显著上调(P<0.001)、IFN-α和IFN-βm RNA的转录水平显著上调(P<0.05),表明IRF7可通过增强下游IFN-I的表达水平抑制BVDV的复制。将pCMV-Flag-IRF7和pCMV-HA-BVDV N^(pro)质粒共转染HEK 293T细胞后,经激光共聚焦试验进一步分析BVDV N^(pro)蛋白与IRF7的相互作用关系,结果显示BVDV N^(pro)蛋白和IRF7蛋白在细胞浆中存在明显的共定位,提示二者可能在细胞浆中存在特异性相互作用。将不同剂量重组质粒pCMV-HA-BVDV N^(pro)分别转染HEK 293T后采用western blot检测,结果显示Npro蛋白可以下调IRF7蛋白的表达,且转染2μg重组质粒组中IRF7蛋白极显著下调(P<0.001)。上述结果首次证实宿主细胞可通过上调IRF7和IFN-I的表达抑制BVDV复制,BVDV的Npro蛋白与IRF7可发生特异性相互作用,并下调IRF7表达水平以拮抗宿主细胞对BVDV的抑制作用。本实验为进一步研究BVDV N^(pro)蛋白的功能奠定基础,也为BVD的防控�Our research delved into the intricate interplay between interferon regulatory factor 7(IRF7)and bovine viral diarrhea virus(BVDV)replication within host cells.We embarked on this investigation by amplifying the BVDV N^(pro) and IRF7 genes via reverse transcription-polymerase chain reaction(RT-PCR),subsequently crafting the eukaryotic expression plasmids pCMV-Flag-IRF7 and pCMV-HA-BVDV N^(pro).These constructs were then transfected to bovine testis(BT)cells,followed by BVDV infection to assess the expression levels of BVDV E2 protein,IRF7,as well as changes in BVDV 5'UTR and IRF7 mRNA transcript levels.Our findings unveiled a profound suppression of BVDV E2 protein expression(P<0.001)and a marked decrease in BVDV 5'UTR transcript levels(P<0.001)in IRF7-overexpressing BT cells post-infection.Notably,IRF7 protein levels were significantly downregulated upon BVDV infection in these cells(P<0.001),whereas IRF7 mRNA transcription levels remained unchanged,suggesting post-transcriptional regulation.These results underscores the inhibitory role of IRF7 overexpression in BVDV replication and its subsequent modulation of IRF7 protein abundance.To elucidate the underlying molecular mechanisms,we analyzed the mRNA transcription levels of IRF7,IFN-α,and IFN-βin IRF7-overexpressing BT cells infected with BVDV at an MOI of 0.1 for 24 hours.Our results demonstrated a significant upregulation of IRF7,IFN-α,and IFN-βmRNA transcripts levels in BVDV-infected IRF7-overexpressing BT cells compared to BVDV-infected control BT cells(P<0.001 for IRF7,P<0.05 for IFN-αand IFN-β).These findings indicate that IRF7 potentiates the antiviral response by enhancing type I interferon(IFN-I)expression,thereby inhibiting BVDV replication.Furthermore,we explored the interaction between BVDV N^(pro) and IRF7 by co-transfection and colocalization assays.The results revealed distinct sub-cellular localization of BVDV N^(pro) and IRF7 proteins in the cytoplasm,suggesting a specific interaction.To corroborate this,we transfected HEK 293T cells wit

关 键 词：牛病毒性腹泻病毒 干扰素 干扰素调节因子7 相互作用 复制 

分 类 号：S855.3[农业科学—临床兽医学]