EV71/CVA16衣壳蛋白VP0的裂解及其对感染性和疫苗免疫原性的影响

Enterovirus 71 (EV71) and Coxsackie A16, a member of the human enterovirus species of the picornavirus family, is a pathogen that has become a serious threat to the health of infants and children. The picornavirus has a single-strand, positive-sense RNA genome, which is packaged by 60 copies of four capsid proteins, VP1, VP2, VP3 and VP4. VP0 is the precursor of both VP2 and VP4, and the cleavage of VP0 to VP2 and VP4 is a key event during the process of particle maturation. However, the mechanism for the VP0 cleavage is still unclear. The structure changes before and after the cleavage and their influences on viral infection remain undetermined. The vaccine stability, immuno-genicity associated with the cleavage also require further investigation. Therefore, we propose to (1) identify the cleavage site of VP0 and to investigate the role of genome RNA in the cleavage; (2) evaluate the effect of the cleavage on viral infectivity through VP0 mutation; (3) analyze the relation between the cleavage and vaccine stability, immunogenicity and protection efficacy. The proposed study will help to understand the process of assembly and maturation of Enterovirus 71(EV71) and Coxsackie A16 and other genus in picornavirus family as well. It is both a basic research to clarify the mechanism of VP0 cleavage and an applied research to guide the rational design of vaccine and fulfill the quality control of vaccine production. Most importantly, it will contribute to the prevention of the HFMD epidemic

手足口病已成为威胁婴幼儿健康的重大传染疾病，EV71和CVA16是手足口病的主要病原体，该病毒为小RNA病毒科肠道病毒属。病毒衣壳由4种结构蛋白组成（VP1-VP4），其中VP4和VP2由一个共同的前体VP0裂解而来。这一裂解过程是病毒衣壳成熟的核心事件，EV71和CVA16 VP0裂解机制未见报道；裂解前后对衣壳结构的变化以及对病毒感染性的影响尚不明确；另外，裂解对疫苗稳定性、免疫原性的作用仍需深入研究。本课题以EV71和CVA16为研究对象，拟寻找并鉴定VP0裂解位点，研究病毒基因组RNA在裂解过程中的作用；通过VP0裂解位点特异性突变明确VP0裂解对病毒感染性的影响；探究VP0裂解在疫苗稳定性、免疫原性和保护性中发挥的作用。本项目既是基础理论研究的深入，同时也是应用基础研究，为疫苗的设计、质量控制提供了理论基础，对手足口病的防治具有科学意义。

手足口病已成为威胁婴幼儿健康的重大传染疾病，EV71和CVA16是手足口病的主要病原体 ，该病毒为小RNA病毒科肠道病毒属。病毒衣壳由4种结构蛋白组成(VP1-VP4)，其中VP4和VP2由一个共同的前体VP0裂解而来。这一裂解过程是病毒衣壳成熟的核心事件，EV71 VP0裂解机制未见报道；VP0裂解前后及VP0的蛋白质修饰对病毒感染性的影响尚不明确。本项目以EV71为研究对象，鉴定了VP0裂解位点位于69K/70S，通过系列突变研究证实70S位点对EV71的组装和感染有较大影响；进一步证明了EV71蛋白酶2A和3C不参与VP0的裂解。项目鉴定了VP0 2位Gly的豆蔻酰化修饰对EV71感染的影响；豆蔻酰化修饰不影响衣壳的组装，但豆蔻酰化修饰的缺失降低EV71的感染性；其机制是EV71感染早期的内吞事件中，衣壳豆蔻酰基团具有与细胞内囊泡膜结构相互作用从而介导脱衣壳的功能。项目还开展了基于EV71 C4基因亚型的手足口病疫苗对EV71各基因型广谱保护性的研究，结果表明EV71 C4基因亚型疫苗免疫的大鼠和婴儿血清均具有广谱交叉中和活性。本项目既是EV71组装与感染机制等基础理论研究的深入，同时也是应用基础研究，为手足口病疫苗的优化提供指导。