新城疫病毒V蛋白靶向MAVS抑制先天性免疫机制研究

Newcastle disease is still one of the most severe infectious diseases that threatening the poultry industry. Newcastle disease virus (NDV) effectively replicates in avian and tumor cells, indicating that it establishes an infection through counteracting the antiviral responses elicited by the cell. The concrete mechanism is not clarified so far. Mitochondrial Antiviral Signaling protein (MAVS) plays a key role in RIG-I-like-receptor-mediated innate immunity. However, currently, there are no reports of NDV, or even any other paramyxovirus, targeting MAVS for inhibition of innate immunity. Our previous results showed that NDV infection induces MAVS degradation at the late stage of infection. V protein plays an important role in MAVS degradation. Therefore, we deduce that V protein disrupts innate immunity by degrading MAVS. First, the key amino acid sites determining MAVS degradation by V protein will be verified by segmented interaction and site mutation. The specific E3 ubiquitin ligase involving in V-induced MAVS degradation will be verified. Then we'll study whether V-induced MAVS degradation affects IFN production and NDV infection. Finally, we test whether V protein of other paramyxovirus has the similar function. Sequence alignment and site mutation are employed to explore the conserved amino acid sites in V protein which mediate MAVS degradation. The present study provides new insights into how NDV and other paramyxovirus inhibit host innate immunity.

新城疫仍是危害养禽业最大的禽病之一。新城疫病毒（NDV）能够特异性的在禽细胞和肿瘤细胞中高效复制，意味着NDV成功阻断或逃逸细胞的抗病毒机制，但具体机制不清。线粒体抗病毒信号蛋白（MAVS）在RIG-I样受体介导的细胞先天性免疫中起枢纽作用，但目前尚未有NDV或其他副黏病毒靶向MAVS抑制先天性免疫的报道。前期研究发现NDV感染后期能够降解MAVS，V蛋白在其中发挥主要作用。本项目拟在此基础上，通过蛋白互作，点突变，免疫荧光，泛素化免疫共沉淀，荧光素酶报告基因检测等技术，确定V蛋白介导MAVS降解的分子机制和关键位点，证实MAVS的降解对下游IFN-β产生以及NDV复制的影响。并验证其他副黏病毒V蛋白对MAVS的降解，找到影响MAVS降解的副黏病毒V蛋白上的关键位点。该研究首先证实V蛋白靶向MAVS抑制先天性免疫，阐明NDV等副黏病毒抑制宿主先天性免疫的新机制。

新城疫仍是危害养禽业最大的禽病之一，新城疫病毒（NDV）能够在禽细胞和人类肿瘤细胞中高效复制，说明病毒成功阻断或逃逸细胞的抗病毒机制。研究NDV如何抑制宿主细胞抗病毒反应对于阐明NDV的复制特性至关重要。线粒体抗病毒信号蛋白（MAVS）在其介导的先天性免疫中起枢纽作用，在长期的病毒与宿主共存过程中，病毒进化出一系列拮抗MAVS的机制。本项目证实NDV的V蛋白通过招募宿主细胞中的E3泛素连接酶RNF5，靶向MAVS的K362/461氨基酸位点降解MAVS，抑制下游IFN信号通路，促进病毒复制，这是副黏病毒科病毒V蛋白的共性机制，这为在感染性克隆水平改造病毒制备新型疫苗，以及针对副黏病毒的抗病毒药物创制奠定了基础，相关成果发表于Journal of Virology。另外，本项目首次克隆了鹅MAVS基因，并验证其以种属特异性方式发挥抗病毒作用，相关成果发表于Developmental and Comparative Immunology，并获得授权专利一项（一种鹅源线粒体抗病毒信号蛋白及其应用），为禽和水禽的抗病育种奠定了基础。另外，对NDV负调控MAVS介导的干扰素信号通路上游进行延伸研究，发现DDX1-DHX36-DDX21复合物的形成也能介导下游MAVS激活，病毒感染切割DDX21并抑制DDX1-DDX21-DHX36复合物的形成，进而抑制下游MAVS介导的IFN-β及细胞因子的产生，相关成果发表于mBio。