通过新的EV71抑制剂suramin靶点鉴定探索EV71病毒宿主相互作用

Our laboratory identified by chemical genetic screening that the P2X receptor antagonist suramin, an approved drug, inhibits EV71 replication in host cells at an early stage in the life cycle. There are seven P2X receptors, all of which are ligand-gated cationic receptors, which respond to extracellular ATP and are important in neuronal signal transduction. EV71 infection of the Central Nervous System infection causes neurogenic pulmonary edema, cardiopulmonary dysfunction, paralysis, CNS infection and inflamation. The pathophysiological mechanisms leading up to neurological symptoms are not understood. We believe that P2X receptors could play a crucial role in this process by facilitating viral replication at the cellular level and triggering P2X signalling events that lead to cardiopulmonary dysfunction. Our project aims at identifying key factors involved in EV71 entry and EV71 viral-cell interaction through the identification of both suramin viral and cellular target proteins as well as the effect of EV71 infection on cellular signalling through P2X receptors..The project will include the following studies: 1. Analyze which P2X subtype/subtypes are involved in EV71 entry by blocking P2X receptor with chemical probes, P2X subtype specific antibodies, and downregulation of P2X surface expression by siRNA; 2. Analyze whether exogenic over-expression of P2X receptors in non-susceptible cell lines confers EV71 susceptibilityt; 3. Identification of key sites in the viral particle that binds to P2X receptors by selecting resistant mutations in the presence of sub-therapeutic amounts of suramin; 4, Analyze whether suramin inhibits the previously described EV71 receptors SCARB2 and PSGL-1; 5, Determine if EV71 is able to trigger signal transduction events through P2X recepors and whether suramin can prevent virus-induced signalling..This project will identify novel cellular pathways and targets of EV71 infection, notably P2X receptors, and identify the viral counterparts that interact with them. This study is aimed to identify an important missing link to explain how CNS infection by EV71 leads to physiopathological changes in children with severe HFMD disease.

P2X拮抗剂suramin通过抑制EV71病毒进入宿主细胞而抑制EV71感染。P2X受体家族是重要的神经信号转导受体。EV71感染能引发神经源性肺水肿、心肺功能损伤、麻痹以及神经系统感染，可能与P2X受体功能异常或病毒通过P2X感染神经系统有关。我们将鉴定suramin抑制EV71感染的病毒和宿主靶点，来探索EV71感染中病毒和宿主细胞的相互作用。研究包括以下内容：1，测定P2X化合物探针、siRNA和亚型特异抗体对EV71感染的抑制作用，初步确定相关的P2X亚型；2，EV71不易感细胞中外源过量表达P2X能否介导EV71感染；3,耐药突变分析鉴定suramin的病毒靶点；4，suramin能否抑制SCARB-2、PSGL-1介导的EV71感染；5，EV71病毒对宿主细胞靶点蛋白信号转导和生理功能的影响。本项目将鉴定EV71病毒宿主相互作用的关键分子，并为EV71神经致病机理提供线索。

EV71可导致严重的中枢神经系统感染，年幼儿童感染后导致多系统衰减并有致死的可能。目前临床尚无有效治疗药物。因此当下任务是开发出EV71感染临床治疗用药。我们从化合物库中筛选得到了EV71有效抑制剂suramin，可作为临床治疗RSV感染的候选用药进行开发。我们建立了绝对荧光定量PCR方法对病毒基因组进行定量分析，结果发现EV71可抑制多个EV71分离株的感染复制，除此之外，还可抑制人肠道病毒基因亚型A中的所有分离株，以及人肠道病毒基因亚型B中的一个分离株，而对CD亚型无作用。通过小鼠和恒河猴感染模型分析发现，suramin对两种动物感染EV71具有良好的保护性。通过鉴定suramin抑制病毒的作用时相，我们进行了多种方案药物处理，结果发现suramin作用主要发生在感作阶段即病毒的粘附和入侵细胞。为了鉴定得到suramin抗病毒作用的靶点，我们进行了药物浓度压力筛选，结果发现耐药毒株筛选失败，我们推测药物靶点可发生回复突变。我们进一步分析了suramin与EV71衣壳的结合模式，发现作为suramin的同类型磺化物均具有抗EV71能力，而suramin的萘磺酸基团是与EV71衣壳结合的关键，这一发现将促进我们对suramin与EV71-VP1以及与宿主细胞膜表面受体的研究，从而借助于suramin这个工具阐述EV71病毒入侵细胞的分子机制。本研究获得发明专利1项，外文论文3篇。