病毒利用细胞蛋白P60以负调控宿主抗病毒防御的作用机理

Antiviral agents based on cellular defense mechanisms have become a major research thrust in the field of virology in the past few years. Recent studies by the present investigator discovered the virus entry mechanisms and corresponding cellular defense strategies for different families of viruses. Several virus-binding proteins have been identified and investigated systematically. Among them, the cellular protein P60 is of particular significance since it is a common target of a variety of viruses with distinct features. Viruses can enforce the migration of P60 from the nuclei of infected cells. Both knock-down and inhibition of nuclear export of P60 were found, for the first time, to effectively suppress replications of eight viruses from six different families. To date, no related research has been reported in the literature. The present investigator found P60 can down-regulate type I interferon and proinflammatory cytokines. Further study revealed that P60 directly targets the dsRNA- dependent protein kinase (PKR), and might subsequently inhibit the expression of interferon and block viral protein synthesis. The proposed research will explore the underlying molecular mechanisms of virus utilizing the cellular protein P60 to negatively regulate host antiviral defense. The specific objectives are to explor (1). the binding domain and the relationship between P60 and PKR; (2). how the virus utilizes P60 to negatively regulate PKR; (3). the effect of P60 on replication of VSV and HSV-1, and the expression of the cytokines described above using P60-/- stable cell lines. Results from the proposed research will not only provide important scientific knowledge to understand comprehensively cellular defense mechanisms, but will also lay the foundation for designing antagonist for developing effective antivirals.

基于解析细胞防御机理研究抗病毒制剂，是病毒学领域的前瞻论题，也是重要研究方向之一。本课题组在揭示多种病毒侵染及细胞防御机理基础上，发现广谱病毒作用靶位P60。病毒感染可使P60从细胞核迁移出来，而P60易位被阻断或其基因沉默可显著抑制6科类8种病毒的复制。本课题组还发现，P60可下调表达I型干扰素与促炎性细胞因子；进一步研究表明，P60直接靶向蛋白激酶PKR，因此可能抑制干扰素的表达并促进病毒蛋白的合成。本项目以PKR为入手点，深入探讨病毒利用细胞蛋白P60以负调控宿主抗病毒防御的作用机理，主要研究包括1）P60与PKR的互作结合域及修饰关系；2）病毒如何利用P60负调控PKR；3）P60敲除的稳定细胞系中，VSV与HSV-1病毒的复制水平，以及干扰素等细胞因子的表达状况。本项目不仅可为全方位认识细胞防御提供新的科学资料，亦为靶向P60设计有高效抗病毒活性的拮抗物奠定基础。

基于解析细胞防御机理研究抗病毒制剂，是病毒学领域的前瞻论题，也是重要研究方向之一。本课题组在揭示多种病毒侵染及细胞防御机理基础上，发现广谱病毒作用靶位P60。病毒感染可使P60从细胞核迁移出来，靶向RIG-I的k63位泛素化，减少PKR等ISG的表达，负调控干扰素先天免疫通路，因此优化病毒复制。本课题组还发现，P60基因沉默与阻断P60易位则可抑制多种病毒复制，表明P60是具有广谱意义的病毒作用靶位，这突破了DNA与RNA病毒，以及病毒有无囊膜的限制。此外，以PKR为入手点，发现HSV-1病毒蛋白ICP34.5利用P60,促进蛋白磷酸酶PP1a募集以去磷酸化真核启动因子eIF2a,从而有效地进行病毒翻译。P60基因敲除也显著减轻HSV-1感染小鼠的组织损伤、炎症反应以及降低病毒产量。我们进一步靶向P60构建shRNA，发现shRNA处理可抑制NDV与AIV感染宿主鸡胚，达2个数量级。本课题组构建P60模拟蛋白G4-T，体外表达纯化与修饰后，以10 ug/ml用量，直接添加到细胞培养液中使用，可提升7科类病毒滴度1-3个数量级，包括有囊膜的病毒：冠状病毒科猪腹泻病毒PEDV、副粘病毒科新城疫病毒NDV、黄病毒科丙型肝炎病毒HCV、正粘病毒科禽流感病毒AIV、疱疹病毒科单纯疱疹病毒HSV-1、弹状病毒科VSV，以及无囊膜的小RNA病毒科肠道病毒EV71。本课题组参与了核糖体生成因子P60的正式命名，并在病毒学领域率先揭示其优化多种病毒复制的作用机理，为全方位认识细胞防御提供新的科学资料；基于这个新发现的病毒靶位，设计具有广谱意义的抗病毒阻断物，可为研制防御感染的共性策略提供可行性新思路。在Cell Death Disease，Drug Discovery Today，Antiviral Research等期刊发表10篇论文，申报并授权国家发明专利2项，培养5名博士生，硕士生1名。

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