基因III型新城疫病毒强毒株HN蛋白与宿主蛋白互作及调控病毒复制机制研究

Field strains of Newcastle Disease virus (NDV), which undergo genetic mutation on viral genes, often increase the virulence and extend its host range. Therefore, it is crucial to explore the molecular basis and host mechanism for increased virulence of NDV. Our previous study demonstrated that velogenic genotype III NDV HN-mediated immune damage in chicken by intravenous inoculation (i.v.) contribute to the high mortality in SPF chickens. However, it is unclear which host factor contribute to HN-mediated the increased virulence of NDV. In this study, to further understand the pathogenesis of NDV HN genen, we will identify target cells and cytokine, and host protein interacted with HN. The following experiments will be performed. First, we will test binding affinity, fusion promotion and NA activity of HN protein of model virus. Second, we will explore the mechanisms of the cell activation and cytokines secretion of peripheral blood mononuclear cell (PBMC) in vitro separated from SPF chicken using flow cytometry and RRT-PCR. Third, we explore the mechanisms of the cell activation, cytokines secretion, autophagy and viral replication in PBMC separated from SPF chicken infected with NDV i.v or oro-nasal. Fourth, cellular proteins interacting with HN will be identified by LC-MS/MS and analyzed by bioinformatics software, and the interaction of the cellular protein and HN proteins will be confirmed by GST-pull down, co-immunoprecipitation and confocal microscopy assays. It will be carried out to identify the molecular mechanism of activation in targeted cells and factors, and biological significance of crucial mutation in HN on the increased virulence. This study may provide evidence of molecular mechanisms of different pathogenesis of NDV.

新城疫病毒（NDV）野毒株发生遗传变异可增强毒力并扩大宿主范围。前期研究发现基因III型毒力增强株，其HN基因所介导的免疫器官损伤是鸡致死的关键机制。但HN发挥该功能潜在的宿主机制还不清楚。本研究旨在探索病毒感染时与HN互作的宿主蛋白及靶细胞和靶分子，并阐明致病机理的生物学意义。研究内容包括：1)测定病毒亲和力、促融合和NA活性；2)体内感染外周血单个核细胞（PBMC），流式细胞术（FCM）分析细胞动态变化和细胞因子转录水平；3)口鼻或静脉接种途径感染鸡，分析PBMC细胞表型、细胞因子转录，免疫器官中自噬的产生与病毒复制、细胞因子转录水平；4)利用LC-MS/MS全面筛选与HN互作的宿主蛋白，IPA进行生物信息学分析，用Co-IP、GST-pull down和激光共聚焦技术对互作验证；5)对病毒激活宿主细胞、互作的宿主蛋白生物学学意义进行分析，研究将为NDV致病差异机制提供理论依据。

① HN蛋白A494D和E495K双突变协同增强基因III型NDV毒力和致病性。基因III型NDV强毒株JS/7/05/Ch与中等毒力Mukteswar株高度同源，但致病性不同。研究表明强毒株HN蛋白A494D和E495K氨基酸变异是毒力主要决定因素；氨基酸的突变引起HN蛋白结构稳态的改变；强毒株具有更强的红细胞吸附活性、促膜融合活性、F蛋白裂解和溶血活性，且在细胞上具备更强的复制能力；强毒株感染SPF鸡后具有更高水平的体内复制能力，并能够诱导更强的炎性因子应答以及脾脏和胸腺的组织病理损伤。.② HN蛋白与宿主骨架蛋白Vimentin互作调控基因III型NDV强毒株复制和致病性。利用LC-MS/MS技术筛选出与HN蛋白存在差异互作的宿主靶蛋白Vimentin。强毒株JS/7/05/Ch的感染显著诱导Vimentin纤维结构的重排，并显著促进病毒的复制；病毒的复制与Vimentin的重排相互依赖。NDV感染对Vimentin的差异调控在HD11细胞中更加显著，JS/7/05/Ch的感染加剧了细胞的病变和损伤，并诱导更高水平的细胞凋亡和自噬。.③ 基因III型NDV强毒株主要靶细胞为单核巨噬细胞。细胞分选结果表明病毒感染可诱导PBMC中单核细胞的增殖，感染后3天单核细胞的相对病毒含量均超过淋巴细胞占据主导地位。JS/7/05/Ch感染鸡在外周血和组织器官中复制水平更高；对脏器中巨噬细胞亲嗜性更强，存在明显的共定位现象，引起肺脏和脾脏炎性细胞浸润和胸腺T淋巴细胞耗竭，部分巨噬细胞会发生凋亡，淋巴细胞耗竭可能与感染病毒后巨噬细胞发生凋亡或其分泌的细胞因子介导的相关功能有关。.④ HN蛋白变异增强NDV强毒株对单核巨噬细胞的亲嗜性，显著降解免疫器官中Vimentin，引起更强的组织损伤。JS/7/05/Ch能够引起HD11（鸡巨噬细胞系）而不是CEF（鸡胚成纤维细胞）Vimentin重排，重排的Vimentin与病毒复制中心相互重叠。感染鸡体内Vimentin与免疫器官中巨噬细胞共定位，JS/7/05/Ch在法氏囊、脾脏、胸腺和腺胃中诱导Vimentin显著降解，同时增强了法氏囊、胸腺和腺胃中自噬蛋白活化以及脾脏、胸腺和腺胃中凋亡蛋白活化，高通量测序发现差异表达的基因主要富集在血液循环和细胞骨架功能。