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。

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).