PPRV非结构蛋白C抑制RLRs介导的I型IFN信号通路的分子机理研究

The non-structural protein C of Peste des petits ruminants virus (PPRV) plays an important roles in it escaping from host innate immune responses. Our previous studies have found that C protein inhibits the retinoic-acid-inducible gene I (RIG-I)-like receptors (RLRs)-mediated type I IFN signaling pathway, but its molecular mechanism remains unclear. In the present program, the recombinant plasmid of pcDNA3.1-Myc-C that expresses the C protein gene was first constructed. The HEK293T cells will be co-transfected pcDNA3.1-Myc-C and RIG-I CARD (RIG-I N) with RIG-I N induced interferon-β (IFN-β), IFN stimulated response element (ISRE) or nuclear factor kappa-B (NF-κB) to detect the its influence for IFNs signaling pathway using a dual-specific luciferase reporter assay（DLRA）. Subsequently, the DLRA will be also performed to identify the candidate target proteins interacted between C protein and the 7 key signaling molecules, which are RIG-I CARD (RIG-I N), melanoma differentiation associated gene 5 (MDA5), mitochondrial antiviral signaling (MAVS), TRAF family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1), IκB kinase (IKKɛ), interferon regulatory factor (IRF3) and IRF7, in the up-stream and down-stream of the RLRs signaling pathway. Furthermore, The co-immunoprecipitation （Co-IP）will be used to ascertain the target molecules and their regulation mechanism in RLRs-mediated type I IFNs sigmaling pathway. The RT-qPCR experiment will be implemented to analyse the expression of IFN-β, and its downstream interferon stimulated gene 56 (ISG56), ISG15 and C-X-C motif chemokine 10 (CXCL10) at trascriptional levels. Virus rescue of reverse genetics technology will be adopted to generate mutant PPRV Nigeria/75/1_ΔC that lacks expression of C protein. Both wild type and mutant type viruses are infected in HEK293T cells to explore the effect of RLRs-mediated type I IFNs signaling pathway and the levels of viruses replication. In addition, the degree of phosphorylation of IRF3 in the viruses infected host cells are tested by immunofluorescence laser confocal technique to further testify the inhibitory effect of protein C on IFNs production. These findings will clarify the molecular mechanism of the RLRs-mediated type I IFN signaling pathway inhibited by the non-structural protein C of Peste des petits ruminants virus (PPRV) so as to provide theoretical basis for the control of Peste des petits ruminants (PPR).

前期研究发现PPRV C蛋白能抑制天然免疫中RLRs介导的I型干扰素生成，但未知其分子机制。本项目运用双荧光素酶报告基因共转染RIG-IN和C蛋白基因与报告基因IFN-β、ISRE或NF-κB至HEK293T细胞，检测C蛋白对IFNs信号通路的影响；进一步共转染C蛋白基因与IFNs通路中候选靶分子RIG-IN、MDA5、MAVS、TBK1、IKKɛ、IRF3和IRF7筛选其作用靶点；免疫共沉淀确定C蛋白作用的靶分子；RT-qPCR分析C蛋白对IFN-β及其下游因子ISG56/15和CXCL10在转录水平的影响。运用反向遗传学病毒拯救技术构建C蛋白基因沉默的突变病毒，感染宿主细胞研究其对IFNs产生的抑制作用，免疫荧光激光共聚焦检测IRF3磷酸化程度，进一步证明C蛋白对IFNs产生的抑制作用。研究结果将阐明PPRV C蛋白抑制I型IFNs信号通路的分子机理，为PPR防控提供理论依据。

小反刍兽疫病毒(PPRV)能引起绵山羊急性、高度传染性小反刍兽疫，严重威胁养羊业发展。与其他麻疹病毒属病毒一样，PPRV引发的急性传染能诱导宿主产生免疫功能抑制，有利于继发性感染建立并加重其病程。研究已证明副粘病毒科病毒通过阻断STAT/JAK磷酸化抑制IFN诱导作用、抑制干扰素诱导的抗病毒蛋白活性、修饰JAK/STAT通路相关分子三个策略在不同水平上阻断IFN-I信号转导，但PPRV非结构蛋白C相关免疫功能抑制研究较少。.本项目运用qPCR、WB、双荧光素酶报告基因检测分析基本阐明了PPRV C蛋白抑制RLR信号通路中RIG-I介导的IFN-I生成分子机理。PPRV体外感染宿主细胞后，病毒RNA通过与RLRs信号通路上接头分子RIG-I介导的MAVS结合，抑制IFN-β的产生。同时，C蛋白进一步抑制JAK-STAT通路中STAT1磷酸化是其抑制IFN产生抗病毒作用的原因。进一步研究表明C蛋白可显著抑制内、外源性IFN对PPRV、EMCV和VSV复制的抑制作用，具有广谱的抗病毒效应。研究结果对进一步阐明C蛋白介导的天然免疫机制和研制抗病毒药物奠定了基础。运用细胞融合与杂交瘤细胞筛选、间接ELISA抗体筛选技术，制备获得抗PPRV N蛋白单克隆抗体3株、C蛋白2株。WB和IFA检测表明单克隆抗体在1:500-5000倍稀释下能有效结合病毒蛋白，具有较好特异性和灵敏性。运用PCR、SOE-PCR、基因点突变和载体多克隆位点改造等技术构建了PPRV正常感染性全长cDNA克隆pBluescript SK(-)-MCS-PPRV、C基因突变沉默表达的感染性全长cDNA克隆pBluescript SK(-)-MCS-PPRV-△C，并在BSRT7/5和GEEc细胞上优化了重组病毒拯救条件。将正常感染性全长cDNA克隆质粒(4μg)、C基因突变感染性全长cDNA克隆质粒(4μg)分别与辅助质粒pCDNA3.1-N(2μg)、-P(1μg)、-L(1μg)共转染表达T7聚合酶的BSRT7/5细胞，培养2d后裂解收集重组病毒上清，WB检测表明拯救出突变重组病毒，成功建立了PPRV反向遗传学研究体系，为进一步研究PPRV C蛋白抑制IFN-I信号通路的分子机理和新型疫苗研制奠定了基础。.本项目发表SCI论文3篇、中文论文3篇(其中第一标注4篇，第二、第四标注各1篇)，毕业研究生3人。