肠道病毒71型调控内质网相关蛋白质降解（ERAD）促进自身复制的分子机制研究

Enterovirus 71 (EV71) is the major causative pathogen of hand, foot and mouth disease. The lack of understanding of its viral pathogenesis impeded the development of drugs against this virus and the control of EV71 infection. Endoplasmic reticulum (ER)-associated degradation (ERAD) is a universally important process among eukaryotic cells. ERAD is necessary to preserve cell integrity since the accumulation of defective proteins results in diseases associated with neurological dysfunction, cancer, and infections. This process involves recognition of misfolded or misassembled proteins that have been translated in association with ER membranes. Recognition of ERAD substrates leads to their extraction through the ER membrane (retrotranslocation), ubiquitination, and destruction by cytosolic proteasomes. Different viruses can manipulate the ERAD process for promoting their replication, chronic persistent infection and immune evasion. However, how EV71 exploits ERAD machinery and benefit for its own replication is unknown so far. Our preliminary results showed that EV71 could inhibit and promote the degradation of ER lumen glycosylated substrate sonic hedgehog (SHH) and homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (Herp), one molecule of the retrotranslocon of ERAD on ER membrane, respectively; EV71 2C protein could interact with multiple ERAD machinery molecules including Hrd1, Herp and p97; MG132, the inhibitor of proteasomes, could inhibit the replication of EV71, suggesting that EV71 might manipulate the ERAD process to promote its own replication, and the detailed molecular mechanism deserves further study. This project aims to clarify the distinct step of ERAD process, including substrate recognition, retrotranslocation, ubiquitination and proteasomal degradation, where the EV71 exploited for different ERAD lumen substrates degradation, and whether the EV71 2C protein or its precursor proteins promote these substrates degradation through interaction with the above mentioned ERAD molecules. Further, we are going to clarify the specific E2 conjugation enzymes and E3 ligases involved in EV71 induced Herp degradation, and check whether Herp plays different roles in ERAD of different ER lumen substrates; Lastly, we will investigate if inhibiting of the ERAD process could promote EV71 taking advantage of the EDEMosomes for its replication and what is the mechanism. These studies would be helpful to disclose new points and enrich of our understanding of the pathogenesis of EV71, further to direct us finding new potential drug targets against EV71.

肠道病毒71型（EV71）是引起手足口病重症和死亡病例的主要病原体，因其致病机制不清目前尚没有特异性抗病毒药物。不同病毒感染以不同形式调控内质网相关蛋白质降解（ERAD）以利于发生免疫逃逸、促进自身复制或形成慢性感染，但EV71感染调控ERAD的机制及与自身复制的关系目前尚不清楚。我们前期研究发现EV71感染可抑制内质网腔内底物（ERAD-L）的降解，促进ERAD转位复合体分子同型半胱氨酸和内质网应激诱导的泛素样结构域蛋白（Herp） 的降解，EV71 2C蛋白可与多个ERAD分子相互作用，蛋白酶体抑制剂可抑制EV71的复制，但具体分子机制仍不清楚。本研究拟在前期工作的基础上，对EV71分别抑制和促进ERAD-L和Herp降解的分子机制进行深入的研究，以期初步阐明 ERAD与EV71复制的调控关系及机制，进一步补充对EV71致病机制的了解，为发现新的抗EV71 药物的靶标提供理论基础。

肠道病毒71型（EV71）是引起手足口病重症和死亡病例的主要病原体，因其致病机制不清目前尚没有特异性抗病毒药物。不同病毒感染以不同形式调控内质网相关蛋白质降解（ERAD）以利于发生免疫逃逸、促进自身复制或形成慢性感染，但EV71感染调控ERAD的机制及与自身复制的关系尚不清楚。本课题对EV71调控内质网相关蛋白质降解（ERAD）分子机制及其对自身复制的影响进行了研究。研究结果发现EV71感染可以抑制不同类型底物的ERAD，既包括依赖CNX/CRT的糖基化底物，也包括依赖Bip的非糖基化底物；EV71对ERAD的抑制作用主要依靠其编码的病毒蛋白酶2A 和3C；EV71的3C蛋白酶可以导致ERAD通路中主要的泛素偶联酶Ubc6e在多个位点发生裂解，其E2结构域最终从从内质网膜上脱落到胞浆中。EV71 2A蛋白酶可以通过抑制蛋白的翻译而导致ERAD通路的重要分子Herp和VIMP的下调表达。通过siRNA筛选ERAD通路相关分子，我们发现p97在病毒复制过程中具有重要作用。进一步的研究表明在EV71感染的细胞中，p97的分布发生了改变，和病毒蛋白2C发生共定位且被证明有相互作用。它们和其它病毒复制相关分子共同定位于病毒复制复合体中，说明其可能在复制复合体的形成中发挥重要作用。结合其它文献报道的p97在病毒复制过程中的作用，我们认为 p97可能是潜在的广谱抗病毒靶点，拟通过 Trim-Away技术建立细胞内 p97的可逆转的敲减体系，目前已通过免疫羊驼后筛库得到多个针对p97的高亲和力纳米抗体序列，并已表达了相应抗体进行后续实验。本研究揭示了EV71对宿主ERAD途径的调控机制，进一步丰富了病毒与宿主细胞蛋白质质量控制系统相互调控的认识，发现了新的潜在的抗EV71感染靶点，促进了对EV71致病机制的了解。