ERK调控IRES翻译起始机制在EV71感染中作用及抗感染策略研究

Protein translation is an essential cellular activity directed by mRNA. Internal ribosome entry site (IRES), a specific element located internally, can direct ribosomes binding to mRNA and form translation initiation complex without cap-dependent translation factors. IRES was first discovered in picornavirus family. It is well known that many viruses take advantage of IRES for viral protein synthesis and spontaneously inhibit hosts' cap-dependent translation. Recently, It is noted that some special cellular proteins, particularly, inflammatory factors, undergo IRES-directed translation. In the case of infection or other stresses, the cap-dependent translation is shut off, whereas IRES-mediated translation plays crucial role in self-protection of cells. However, the underlining mechanisms of switches between cellular and viral IRESes are not yet understood. Enterovirus 71 (EV71), a member of Picornaviruses, is a neurotropic pathogen that causes severe neural diseases and complications in hand, foot, and mouth disease (HFMD) patients in childhood. We have developed effective MEK1 inhibitors and discovered that block of MEK/ERK pathway by MEK1 inhibitors effectively suppresses the EV71-IRES-directed reporter gene expression under stresses. In this study, we aim to further extend our study to host-virus interactions. We will explore the important role of ERK/MEK signaling in regulating switches viral and cellular IRESes-mediated translation, i.e., IRES-directed viral replication and inflammation responses in stressed cells. We will elucidate how MEK1 inhibitors affect viral replication and IRES-mediated cellular translation, as well as anti-inflammation effect. We will try to prove our hypothesis (referred to as "ERK-IRES" model) that ERK signaling is pivotal not only in IRES-mediated viral replication, but neuropathogenesis as well. Our study will provide new knowledge for better understanding the molecular mechanism(s) involved in EV71 replication and neurological pathogenesis caused by EV71 infection. The output of this study will shed light on developing novel, multiple, comprehensive strategic therapies, which may also be applied to other viral infections. This study meets the principle that basic researches should be in accordance with national demand. Therefore, the outputs of this study have not only theoretical importance, but application potential for public health as well.

目前EV71脑炎发病机制不明。蛋白翻译是生物最基本生命活动。IRES翻译启动机制首先在小RNA病毒中发现，后证明感染应激细胞帽状翻译抑制时，细胞IRES机制也发挥重要作用。目前尚不清楚这两种IRES机制在感染细胞中的关系及机制。我们近期发现，感染应激细胞中抑制ERK通路能有效下调EV71 IRES翻译。本研究拟从IRES机制入手，将病毒和宿主细胞联系，通过研究病毒增殖和细胞炎症应激应答中IRES机制作用及关系，阐明细胞ERK通路在调控病毒和细胞IRESes翻译中重要作用。在此基础上利用前期工作获得的靶向MEK1-ERK通路小分子抑制剂，验证其抑制病毒和细胞IRESes翻译效率及抗病毒抗炎作用。最终确立我们的假说EV71感染细胞"ERK-IRESes模式"重要性，揭示基于该机制的EV71脑炎发病机制及探索新型治疗策略，体现基础研究与国家需求结合原则。因此具有良好理论意义、社会需求及应用前景。

蛋白翻译是生物最基本的生命活动。IRES翻译启动机制首先在小RNA病毒中被发现，后在帽状翻译抑制的细胞中也发现了细胞IRES机制。越来越多的研究证明，ERK通路活性对肠道病毒的增殖是必不可少的，但迄今其机制还未阐明。.我们前期研究发现，病毒感染的细胞中抑制ERK通路能够有效下调EV71 IRES介导的翻译。本项目进一步研究证明细胞ERK通路正向调控EV-A71 2A的酶活性，即ERK通路蛋白可调控EV-A71 2Apro介导对细胞eIF4G蛋白切割。eIF4G蛋白切割后促进了病毒IRES介导的翻译，从而增强了病毒的增殖。这是本研究新发现的ERK通路活性对肠道病毒增殖正向调控的一个重要及关健机制，这一结论为ERK通路成为新型的抗肠道病毒分子靶点奠定了重要理论基础。.在此基础上，本课题设计合成了一系列具有原创性的小分子抑制剂，即香豆素衍生物MEK小分子抑制剂（13，14）以及基于药效团模型虚拟筛选合成的靶向MEK激酶抑制剂化合物（9k、9m）。使用病毒学和细胞学方法评估发现，这两类小分子均具有良好抗病毒作用，而且其抗病毒机制可能与抑制EV71-IRES依赖的翻译起始机制有关。特别是我们近期发现细胞中联合使用小剂量无抗病毒效果的ERK通路抑制剂可不同程度恢复干扰素对EV-A71的抗病毒作用。这说明靶向抑制ERK通路既能够抑制病毒的复制，也能恢复机体固有免疫机能。这提示本课题研制靶向抑制ERK通路的小分子候选药物具有良好的应用前景。.本研究基于宿主ERK信号通路对EV-A71 2Apro-IRES介导的翻译调控作用，丰富了宿主ERK通路-病毒相互作用关系的分子机制，从而为新型抗病毒药物研发奠定了良好的理论和实验基础。因此，本研究获得的结果具有良好的理论意义和实际应用前景，且具有良好的原创性。