H7N9 IAV PB1蛋白抑制Ⅰ型IFN信号通路  

PB1 Protein of H7N9 IAV Inhibits the Type Ⅰ IFN Signaling Pathway

作　　者：王婉冰[1,2] 曾艳[2] 汪亮[2] 韩璐[2] 王正祥[2] 陈化兰[3] 李郁[1] 朱启运[1,2] 

机构地区：[1]安徽农业大学动物科技学院,合肥230036 [2]中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室,兰州730046 [3]中国农业科学院哈尔滨兽医研究所/兽医生物技术国家重点实验室,哈尔滨150069

出　　处：《农业生物技术学报》2017年第7期1188-1196,共9页Journal of Agricultural Biotechnology

基　　金：十三五国家重点研发计划专项(No.2016YFD0500207)

摘　　要：H7N9 A型流感病毒(Influenza A virus,IAV)是对公共卫生安全危害严重的新发人兽共患病病原。为了初步解析H7N9 IAV介导的天然免疫反应的分子机制,本研究将聚合酶碱性蛋白1(polymerase basic protein 1,PB1)基因克隆至p RK真核表达载体,并利用定点突变技术对PB1基因的第96位和第129位核苷酸分别进行定点突变,使其移框编码的PB1-F2基因不表达,排除PB1-F2基因对PB1后续研究的影响。随后,利用双荧光素酶报告基因系统和q RT-PCR分析PB1蛋白对Ⅰ型干扰素(interferons,IFNs)信号通路活化的影响;进而,通过免疫共沉淀实验确定PB1蛋白在维甲酸诱导基因Ⅰ样受体(retinoic-acidinducible geneⅠ(RIG-Ⅰ)-like receptors,RLRs)信号通路中的作用靶点,最后在293T细胞中分别过表达PB1蛋白和线粒体抗病毒信号分子(mitochondrial antiviral signaling,MAVS)或RIG-Ⅰ,检测PB1对MAVS或RIG-Ⅰ表达量的影响。结果表明,PB1可以显著抑制IFN-β、干扰素刺激反应元件(IFN stimulated response element,ISRE)和核调节因子-κB(nuclear factor kappa-B,NF-κB)报告基因的活性,并下调IFN-β和干扰素诱导基因56(interferon stimulated gene 56,ISG56)、ISG15和CXC趋化因子配体10基因(C-X-C motif chemokine 10,CXCL-10)的表达;PB1可以特异性地和RLRs信号通路中的MAVS相互作用,并且随着PB1蛋白表达的增加,MAVS的表达呈下降趋势,但RIG-Ⅰ的表达不受影响。因此,本研究的实验数据初步表明,H7N9 PB1蛋白可特异性结合RLRs信号通路中重要接头分子MAVS,并能够降低其表达量,从而抑制Ⅰ型IFN基因的表达并阻断其发挥作用的下游信号通路。本研究结果明确了H7N9 IAV PB1蛋白在天然免疫信号通路中的作用靶点,为防控H7N9流感提供理论依据。H7N9 Influenza A virus （IAV） is an important pathogen of zoonosis health of human. In the present study, to investigate the H7N9 IAV-mediated recombinant plasmids expressing the polymerase basic protein 1 （PB1） gene were to exclude the influence of PB1-F2 that is encoded by PB1 via a frame-shifting which threatens the public innate immune responses, first constructed. However, manner, two nucleotides at positions of 96 and 129 in PB1 gene were mutated by site-directed mutagenesis, respectively. Subsequently, the results from a dual-specific luciferase reporter assay indicated that PB1 significantly inhibited retinoic- acid-inducible gene I CARD （RIG- I N）-induced interferon-[3 （1FN-fl）, type I interferon （IFN） stimulated response element （ISRE） and nuclear factor kappa-B （NF-KB） activities. Consistently, the qRT-PCR experiment confirmed that the expression of 1FN-~ and its downstream interferon stimulated gene 56 （ISG56） and ISG15, and C-X-C motif chemokine 10 （CXCLIO） were down-regulated at the transcriptional levels, suggesting PB1 inhibits the RIG- I -mediated signaling pathway. Furthermore, the co-immunoprecipitation experiment was performed to test the interaction between PB 1 with the key molecules in the RIG- I signaling pathway. The results indicated that H7N9 PB1 specifically interacted with mitochondrial antiviral signaling （MAVS）. In addition, PB 1 was co-transfected dose-dependently with RIG- I or MAVS into HEK293 cells, the Western blotting data showed that the increased expression of H7N9 PB 1 inhibited MAVS expression, but not RIG- I. Therefore, the preliminary data in the present study demonstrated that H7N9 PB1 was specifically associated with the important adaptor, MAVS in the RLRs signaling pathway and decreased the MAVS expression, leading to the inhibition of IFNs expression and the blockade of the downstream signaling transduction. Our finding revealed that the target of H7N9 IAV PB1-associating in the RLRs signaling pathway would be helpful for the H7

关 键 词：H7N9 聚合酶碱性蛋白1(PB1) Ⅰ型干扰素(IFNs) 线粒体抗病毒信号分子(MAVS) 

分 类 号：S855.3[农业科学—临床兽医学]