磷酸化修饰对肠道病毒71型非结构蛋白2C的调控机制研究

Human enteroviruses are a group of positive-stranded RNA viruses, which belong to the family Picornaviridae that contains numerous important human and animal pathogens, including poliovirus (PV), enterovirus 71 (EV71), Coxsackie A virus (CAV), and Coxsackie B virus (CBV), causes around 3 billion human infections annually, responsible for a wide spectrum of diseases, ranging from relatively mild symptoms such as common cold, upper respiratory illness, and hand-foot-and-mouth disease (HFMD), to severe ones such as aseptic meningitis, encephalitis, myocarditis, neonatal sepsis-like disease, and poliomyelitis. So far, although vaccination has almost eradicated poliomyelitis around the world, no effective antiviral therapy is available for many other important human enteroviruses, particularly neuron-tropic EV71 that is the major causative pathogen for HFMD and a serious lethal threat to children. Enterovirus encodes a single polyprotein that is proteolytically cleaved into separate structural and nonstructural proteins. Among them, 2C is the most conserved but least understood one. Enterovirus 2C has long been predicted as a putative superfamily 3 (SF3) helicase on the basis of its AAA+ ATPase activity and conserved SF3 signature motifs. Our previous study has reported that eukaryotically expressed EV71 2C is able to function not only as an RNA helicase that unwinds RNA helices ATP-dependently, but also as an RNA chaperone that ATP-independently destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation. Moreover, we determined the domain requirements for these two RNA remodeling activities associated with 2C, and showed that protein 2C facilitates the EV71 RNA-dependent RNA polymerase (RdRP)-mediated synthesis of (+)-vRNA from (-)-vRNA template in vitro, and when the helicase activity of 2C being disrupted by point mutation, the RNA replication and virus production of EV71 were mostly abolished in cells. These data indicate that 2C-mediated RNA remodeling plays critical roles in enteroviral life cycle, particularly enteroviral replication. In addition, our data show that the RNA helicase and chaperoning activities are also conserved in CAV 2C. Interestingly, we found that although both the prokaryotically and eukaryotically expressed 2C proteins possessed similar ATPase activity, only the eukaryotically expressed 2C possessed the RNA helix unwinding activity. Moreover, the eukaryotically expressed 2C showed an apparently higher molecular mass than the prokaryotically expressed one, implying that the former protein contains some post-translational modifications (PTMs), which potentially play critical role(s) in the functions of 2C. Our further preliminary studies revealed that eukaryotically expressed 2C is modified by phosphorylation, and when being dephosphorylated by phosphatase treatment, this protein lost almost all its helix unwinding activity. These preliminary data indicates that phosphorylation plays an indispensable role in the proper function of 2C. In this project, we aim to use the biochemistry, molecular biology, proteomics and virology strategies to identify the phosphorylation sites in EV71 2C, and further elucidate the regulatory roles of the phosphorylation to 2C as well as the infection, replication and immune evasion of EV71. In addition, we plan to identify the kinase(s) responsible for the phosphorylation of 2C, and further study the roles of the kinase(s) and relevant signaling pathway(s) to EV71 and other enteroviruses. The successful accomplishment of this project will foster the understanding to EV71 and other enteroviruses, and probably provide novel targets for anti-EV71 therapy.

肠道病毒71型（EV71）是导致婴幼儿手足口病以及神经系统并发症甚至死亡的主要病原。2C是肠道病毒中最保守的非结构蛋白。申请人已发表的研究表明EV71及其他肠道病毒2C具有RNA解旋酶及分子伴侣活性，在病毒RNA复制中发挥关键作用。我们的前期研究进一步发现，2C蛋白具有磷酸化修饰，且该修饰对其功能至关重要。在本项目中，我们拟通过一系列生化、分子、蛋白质组学、病毒学手段，鉴定EV71 2C的磷酸化，确定影响2C功能的关键磷酸化位点，深入研究磷酸化对EV71感染、复制及免疫逃逸的影响及机制。在此基础上，我们将进一步筛选细胞内参与2C磷酸化的磷酸激酶及信号通路，研究相关磷酸激酶与信号通路对EV71及其他肠道病毒的调控机制。本项目的成功进行将加深我们对以EV71为代表的肠道病毒的复制及致病机制的认识，拓展我们对病毒与宿主相互作用的理解，并为肠道病毒的抗病毒疗法提供新的靶标及理论基础。

肠道病毒属于小RNA病毒科（Picornaviridae）肠道病毒属（Enterovirus），是重要的导致人类疾病的病毒。据报道，肠道病毒每年在全世界范围内感染约30亿人次，造成了包括脊髓灰质炎、手足口病、普通感冒、出血性角膜炎、病毒性心肌炎等多种疾病，严重危害人类健康。其中，肠道病毒71型（EV71）是导致婴幼儿手足口病以及神经系统并发症甚至死亡的主要病原。2C是肠道病毒中最保守的非结构蛋白，而且在多种其他病毒也编码有与2C同源的2C类似蛋白（如杯状病毒科的诺如病毒所编码的NS3蛋白）。本项目负责人前期已发表的研究表明EV71及其他肠道病毒2C具有RNA解旋酶及分子伴侣活性，在病毒RNA复制中发挥关键作用。因此，对肠道病毒非结构蛋白2C的功能与翻译后修饰调控机制的研究，将有助于加深我们对以EV71为代表的肠道病毒的复制及致病机制的认识，拓展我们对病毒与宿主相互作用的理解，并为肠道病毒的抗病毒疗法提供新的靶标及理论基础。在本项目支持下，我们发现EV71 2C能与宿主蛋白ARRDC3相互作用，并发现ARRDC3能招募一种宿主E3连接酶，对2C泛素化，从而影响2C在EV71感染细胞内的稳定性及功能，从而对EV71病毒的复制产生显著的抑制作用。同时，我们通过理性设计与药物筛选，研发了针对2C蛋白解旋酶活性的抑制多肽，该多肽能在体外水平抑制2C的解旋酶活性，并在细胞与动物水平显著抑制EV71及其他肠道病毒的复制，具有很好的抗病毒效果，有望进一步开发为广谱抗肠道病毒药物。该项工作已完成，已申请发明专利一项（见附件），相关论文正在投稿并修回中。此外，我们还发现了EV71的非结构蛋白3A在逃逸RNAi介导的抗病毒免疫中的关键作用；发现了诺如病毒、埃博拉病毒、新型冠状病毒所编码的解旋酶，对其功能进行了解析，并发现了其潜在抗病毒药物。同时，在本项目支持下，我们也在新型冠状病毒及新冠肺炎致病机制研究上取得了一系列重要研究成果，在包括Immunity、National Science Review、Science Advances、Nucleic Acids Res.、Cell Mol Immunol.等高水平期刊发表。这些工作拓展了我们对病毒与宿主相互作用的理解，揭示了肠道病毒的新的抗病毒药物靶标，体现了国家自然科学基金对自由探索与重要科研产出的支持。