基于ERK/p38-eIF4E通路探讨PEDV抗病毒制剂的作用机理

Antiviral agents based on host cell-virus interaction mechanisms have become a major research thrust in the field of virology in the past few years. Viruses must utilize the cellular translation machinery to produce viral proteins. Viral infection can activate ERK/p38-eIF4E pathway; phospho-eIF4E thus mediates 5’-cap dependent translation initiation. Porcine epidemic diarrhea coronavirus (PEDV) has severely threatened swine industry. There is no specific drug available for the treatment of PEDV infection. Based on the functional screen, we found homoharringtonine (HHT), which targets and degrades the phospho-eIF4E, markedly inhibits viral replication. It is suggested that HHT prevents eIF4E-dependent translation machinery against viral manipulation. The proposed research attempts to exploit the mechanism in which HHT inhibits PEDV infection. The specific objectives are to explore (1). the role of ERK/p38-eIF4E pathway in PEDV replication; (2). how the HHT regulates eIF4E modification to selectively inhibit viral replication, by using puromycin labeling, protein modification analysis and regression testing; (3). in which the stage of viral infection the HHT plays a role and the antiviral activity of HHT in vitro and in vivo. Results from the proposed research will not only provide important scientific knowledge to understand the host cell-virus interaction, but will also lay the foundation for the development of antiviral agents against PEDV infection.

基于解析细胞-病毒互作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。许多病毒感染都可激活ERK/p38-eIF4E通路，被磷酸化的eIF4E继而启动帽子结构依赖性翻译，利于病毒自身蛋白的产生。猪流行性腹泻病毒PEDV给养猪业造成严重损失，目前尚无特效防治药物。本课题组基于功能学筛选，发现磷酸化eIF4E的抑制物——高三尖杉酯碱HHT具有高效的抗PEDV活性，推测HHT通过阻断病毒利用细胞翻译系统，因此抑制病毒复制。本项目深入探讨HHT抗病毒作用机理，主要内容包括：1）ERK/p38-eIF4E通路在PEDV复制中的作用；2）嘌呤霉素标记、蛋白修饰分析与回归试验，探究HHT如何靶向调节eIF4E的翻译后修饰，选择性抑制病毒复制；3）HHT抗病毒作用阶段以及体内外活性验证。本项目为认识病毒与细胞的互作关系提供了新的科学资料，亦为开发仔猪病毒性腹泻药物提供依据。

基于解析细胞-病毒互作机理研究抗病毒制剂，是病毒学领域重要研究方向之一。本课题发现高三尖杉酯碱HHT—作为p-eIF4E抑制子，具有高效的抗感染活性，以及广谱的抗病毒效果，动物实验效果显著。我们还将HHT与氯喹等联合用药，效果更加明显。病毒感染过程中会激活ERK/p38-eIF4E细胞通路，提高p-eIF4E水平；p-eIF4E有利于病毒自身蛋白的产生，因此是抗病毒制剂的有效靶位。此外，我们还研究了p-4E抑制剂RAF265的作用机理及抑制效果，以及基于p-eIF4E进行TMT定量蛋白质组筛选，这些结果为进一步了解p-eIF4E在病毒复制中的意义提供进一步资料。本项目为开发仔猪病毒性腹泻药物提供依据。