2群坦布苏病毒NS1蛋白抑制鸭Ⅰ型干扰素产生的机制研究  

Research on NS1 Proteins of Group 2 Tembusu Virus Inhibiting the Production of Duck Type I IFN

作　　者：黄允真 张俊勤 李林林 董嘉文 向勇 廖明 孙敏华 HUANG Yunzhen;ZHANG Junqin;LI Linlin;DONG Jiawen;XIANG Yong;LIAO Ming;SUN Minhua(Institute of Animal Health,Guangdong Academy of Agricultural Sciences/Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Diseases,Ministry of Agriculture and Rural Affairs/Key Laboratory of Livestock Disease Prevention of Guangdong Province,Guangzhou 510640,China)

机构地区：[1]广东省农业科学院动物卫生研究所/农业农村部禽流感等家禽重大疾病防控重点实验室/广东省畜禽疫病防治研究重点实验室,广东广州510640

出　　处：《广东农业科学》2024年第7期151-160,共10页Guangdong Agricultural Sciences

基　　金：国家自然科学基金(32102691,31902272);广东省科技计划项目(2021B1212030015);广东省“十四五”农业科技创新十大主攻方向“揭榜挂帅”项目(2022SDZG02);广东省现代农业产业技术体系创新团队项目(2023KJ137)。

摘　　要：【目的】坦布苏病毒(Tembusu virus,TMUV)病是重要的水禽传染病,研究发现TMUV感染鸭早期,2群TMUV在肝、肾、脑等组织中的病毒拷贝数显著高于3群TMUV。研究2群TMUV非结构蛋白(Non-structural protein,NS protein)和3群TMUV NS蛋白对鸭天然免疫反应是否存在差异。【方法】以2群TMUV-JM株和3群TMUV-GX株为研究对象,构建两种毒株NS蛋白真核表达质粒,通过双荧光素酶报告系统研究NS蛋白对视黄酸诱导基因蛋白I(Retinoic acid-inducible gene protein I,RIG-I)诱导的β干扰素(βinterferon,IFN-β,为I型)启动子活性的影响。构建重组嵌合NS1蛋白真核表达质粒,通过激光共聚焦、免疫共沉淀、Western Blotting技术研究NS1与RIG-I信号通路中关键分子的互作区域。利用反向遗传操作系统构建NS1重组嵌合病毒,研究NS1在TMUV感染DEF细胞后对IFN-β产生的抑制作用。【结果】TMUV-JM株NS1能抑制RIG-I诱导的IFN-β启动子活性,而TMUV-GX株NS1对其无抑制作用。进一步研究发现,TANK结合激酶1(TANK-binding kinase 1,TBK1)为TMUV-JM NS1蛋白抑制RIG-I介导的I型IFN表达的作用节点分子,且NS1蛋白255~352 aa为发挥抑制作用的功能区域。TMUV-JM NS1与TBK1不存在直接相互作用,是间接降低TBK1磷酸化水平从而降低I型IFN的表达量。【结论】2群TMUV NS1具有抑制RIG-I信号通路的活性,并鉴定出其抑制功能的关键活性区域及作用的通路节点分子,明确2群TMUV NS1通过间接抑制TBK1磷酸化而影响鸭I型IFN产生。【Objective】Tembusu virus(TMUV)disease is an important infectious disease in waterfowl.In previous studies,we found that in the early stages of TMUV infection in ducks,the viral copy number of group 2 TMUV in organs such as the liver,kidney,and brain were significantly higher than those of group 3 TMUV.The study aimed to explore whether group 2 TMUV NS proteins had different effects on the innate immune response of ducks compared with group 3 TMUV.【Method】Taking the group 2 TMUV-JM strain and the group 3 TMUV-GX strain as research subjects,we constructed the eukaryotic expression plasmids of NS proteins of the two strains and compared the effects of the NS proteins on the RIG-I-induced activity of the IFN-β(Type I)promoter by a dual-Luciferase reporter system.With the eukaryotic expression plasmids of the constructed recombinant chimeric NS1 proteins,we investigated the interaction region between NS1 and the key molecules of RIG-I signaling pathway by confocal laser scanning microscopy,co-IP and Western Blotting.By using the reverse genetic system,we constructed NS1 recombinant chimeric TMUV to clarify the inhibitory effect of NS1 on the IFN-βin DEF cells with TMUV infection.【Result】The study results showed that TMUV-JM NS1 could inhibit RIG-I-induced activation of the IFN-βpromoter,while TMUV-GX NS1 did not exhibit the inhibitory activity.Further studies revealed that the TMUV-JM NS1 inhibited the expression of type I IFN via targeting TBK1,and the NS1255-352 aa was the functional region of the inhibitory effect.It was found that TMUV-JM NS1 did not interact with TBK1 directly,but reduced the phosphorylation level of TBK1 indirectly,thereby suppressed the expression of type I IFN.【Conclusion】Based on the study results,we found that the group 2 TMUV NS1 has activity in inhibiting the RIG-I signaling pathway,identified the key activity region and targeted molecule of its inhibitory pathway,and clarified that group 2 TMUV NS1 affected duck type I IFN production by indirectly inhibiting the phospho

关 键 词：坦布苏病毒 非结构蛋白 RIG-I信号通路 I型干扰素 TBK1磷酸化 

分 类 号：S855.3[农业科学—临床兽医学]