SUMO化修饰对EV71 3D蛋白RNA聚合酶活性及病毒复制与致病性的影响

In the past few years, morbidity, especially severe neurological syndromes,which is associated with EV71 has been increasing in China.Understanding of EV71 pathogenisis is crucial for the disease prevention.3D is an RNA dependent RNA polymerase (RdRp)responsible for EV71 genomic RNA replication.The lack of 3'-5' proofreading activity leads to relatively rapid variation and evolution of the virus.Therefore, 3D could be selected as a major antiviral target.SUMOylation is one of the post-translational modifications of proteins.SUMOylation of a protein has many consequences, including alteration of the target protein's stability and biological functions. This may be employed by virues to complete their life cycle.Our previous work confirmed that 3D was SUMOylated when overexpressed.We wish to locate the specific amino acid residue(s) on 3D that determine its SUMOylation.Next,we will creat mutants containing these amino acids to test if RdRp activity is affected.Then we will construct EV71 infectious clones with these amino acid mutations, and compare the viral replication capacity at cellular level and in suckling mice.Our study will help better understand the influence of 3D SUMOylation on EV71 replication and pathogenicity.It may also provide new antiviral tagets for EV71 prevention,and critical information of the relationship between SUMOylation and RNA virus life cycle.

近年来，我国EV71的发病率尤其是重症发病率呈上升趋势，研究其致病机理对其预防和控制显得尤为重要。EV71编码的RNA聚合酶3D负责病毒基因组复制，它的低保真度对病毒的快速变异与进化起直接作用，因此3D可作为抗病毒药物研究的重要靶标。SUMO化作为蛋白翻译后修饰的一种方式，可以调控蛋白质的稳定性和生物学功能，对病毒的复制至关重要。我们的前期工作证明EV71 3D可被SUMO化。本项目将首先鉴定3D蛋白SUMO化的关键位点，并确定SUMO修饰对3D聚合酶活性的影响；然后通过感染性克隆构建3D蛋白SUMO化位点突变的重组病毒；最后分别在细胞和动物水平比较突变前后病毒复制能力及致病性的变化，揭示3D蛋白SUMO化对EV71复制和致病性的影响。本研究有助于深入了解EV71的复制机制，为探索SUMO修饰与RNA病毒生活周期之间的关系提供重要信息，也为EV71的防治提供新靶标。

越来越多的病毒被报道能与宿主的蛋白进行相互作用，包括宿主的翻译后修饰系统，病毒利用宿主的修饰系统以逃逸宿主的免疫。肠道病毒71型（EV71）作为引起手足口病的主要病原，能感染3岁以下儿童病引起严重的神经系统感染和脑膜炎。目前，针对该病毒并没有特效治疗的药物，通过研究该病毒在复制过程中与宿主的相互作用，特别是翻译后修饰对病毒的聚合酶的影响，能进一步的了解病毒的复制机制。在本课题中，通过病毒水平和体外SUMO化修饰系统，我们发现病毒编码的RNA依赖的RNA聚合酶3D蛋白能被SUMO-1修饰。通过定点突变，发现K159和L150/D151/L152是3D的SUMO化修饰的位点。通过体外酶活实验，发现突变3D的SUMO化修饰位点会降低3D的酶活。放线菌酮的蛋白半衰期实验发现3D蛋白的泛素化修饰依赖于SUMO化修饰，二者共同起到稳定3D蛋白的作用。用EV71病毒感染过量表达SUMO-1的细胞，发现3D的泛素化修饰和SUMO化修饰水平都增加了，并且病毒的复制也提高了，推测3D的SUMO化修饰可能是SUMO-Ub的嵌合修饰，二者起到稳定3D的作用，最终起到促进病毒复制的效果。本研究有助于深入了解EV71的复制机制，请为EV71的防治提供新的靶标。