与猪瘟兔化弱毒疫苗株E2蛋白相互作用的家兔脾细胞蛋白的筛选和功能鉴定

Interactions between viral and host proteins are involved in viral infection and pathogenicity. There are distinctive differences between the highly virulent strain and C-strain, a lapinized attenuated vaccine of classical swine fever virus (CSFV) in terms of their replication and pathogenicity in pigs and rabbits. Our previous study indicates that the coding region of C-strain genome is essential for its replication in the spleen of rabbits. However, it remains unclear how host proteins are playing their roles in the adaptation of C-strain to the rabbit. The E2 protein of CSFV is responsible for tissue (cell) tropism and mediating viral entry into cells. This proposal aims to: (1) screen and identify splenic proteins that exclusively interact with the C-strain E2 protein by GST pulldown, co-immunoprecipitation, co-localization, and in situ proximity ligation assays; and (2) investigate functional aspects of identified cellular proteins in the viral replication of C-strain by overexpression/knockdown, and antibody/soluble protein blocking assays. In addition, a series of E2 chimeras containing the partial region(s) of C-strain based on the Shimen strain backbone will be constructed to verify the key region(s) or site(s) responsible for the interactions between the cellular proteins and the C-strain E2 protein. This project is expected to uncover the host cellular proteins and their regulatory roles that are critical for the differences in phenotypes between the highly virulent strain and C-strain of CSFV, and provide scientific data for elucidating mechanisms underlying the C-strain adaptation to the rabbit.

病毒的感染和致病性通常与病毒蛋白与宿主蛋白的相互作用有关。猪瘟病毒强毒株和猪瘟兔化弱毒疫苗株（C株）体内外增殖特性以及对猪和家兔的致病性等方面存在明显差异。我们前期的研究发现，C株适应家兔及其在脾脏复制的主要决定区位于病毒基因组的编码区，但对于宿主在C株适应家兔中扮演的作用并不清楚。鉴于猪瘟病毒E2蛋白决定其组织（细胞）嗜性并介导病毒侵入宿主，本研究拟利用GST pulldown、免疫共沉淀、激光共聚焦、原位邻位连接试验等技术筛选和鉴定与C株E2蛋白专一性相互作用的家兔脾细胞蛋白，通过上调和下调表达、抗体封闭试验、可溶性蛋白阻断试验等研究宿主蛋白在C株复制中的作用。同时，通过构建强毒株和C株E2蛋白的若干嵌合突变体，确定C株E2蛋白上与兔脾细胞蛋白相互作用的关键区域或位点。本项目有望揭示与猪瘟强弱毒表型差异相关的关键细胞蛋白及其病毒调控作用，并为阐明C株适应家兔的分子机制提供有价值的数据。

病毒的感染和致病性通常与病毒蛋白与宿主蛋白的相互作用有关。猪瘟病毒野毒（强毒株）和猪瘟兔化弱毒疫苗株（C株）体内外增殖特性以及对猪和家兔的致病性等方面存在明显差异。我们前期的研究发现，C株适应家兔及其在脾脏复制的主要决定区位于病毒基因组的编码区，但对于宿主在C株适应家兔中扮演的作用并不清楚。鉴于猪瘟病毒E2蛋白决定其组织（细胞）嗜性并介导病毒侵入宿主，本研究通过酵母双杂交技术筛选了多种与猪瘟强弱毒E2蛋白相互作用的家兔脾细胞蛋白，利用酵母共转化试验、GST pulldown试验、共聚焦试验和原位邻位连接试验等证实了硫氧还蛋白2（Trx2）与CSFV 强毒E2蛋白的相互作用，并证实Trx2呈剂量依赖性地激活宿主的抗病毒NF-κB信号通路，而Trx2通过促进NF-κB的p65亚单位入核从而激活该通路。另外证实了丝裂原活化的蛋白激酶激酶2（MEK2）与E2蛋白的相互作用，MEK2且呈剂量依赖性地抑制宿主的抗病毒IFN-β信号通路，从而促进病毒的复制。以上研究从宿主角度，鉴定出猪瘟强、弱毒表型差异的关键宿主因子。另外，从病毒角度，对野毒株和疫苗株（C株）的差异进行了扩展研究，基于流行病学研究从多地C株疫苗免疫场分离获得新的基因亚型（2.1d）猪瘟病毒，发现其不能被C株诱导的抗体有效中和，同时C株疫苗不能对流行毒株提供完全的免疫保护，提示E2蛋白的变异可能导致病毒逃逸疫苗（C株）抗体介导的免疫保护。这为进一步研究猪瘟野毒和疫苗C株差异的分子机制奠定了基础。本项目揭示了与猪瘟强弱毒表型差异相关的关键细胞蛋白及其病毒调控作用，为阐明C猪瘟野毒和疫苗株表型差异的分子机制提供了有价值的数据。