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学科主题生物物理学
埃博拉病毒表面糖蛋白GP与中和抗体复合物电镜结构研究
桂淼
2016-12-28
导师尹长城
专业生物物理学
授予单位北京大学
授予地点北京大学基础医学院
学位博士
关键词埃博拉病毒 GP 中和抗体 冷冻电镜 病毒膜融合
其他题名Electron Microscopy Study of the Ebola Virus Glycoprotein and Neutralizing Antibody Complex
分类号R373.3
摘要

埃博拉病毒(Ebola Virus, EBOV)是一种反义、单链RNA病毒,其表面被覆包膜(Envelope),属于线状病毒科(Filoviridae)。1976年在非洲首次发现后,埃博拉病毒曾出现过多次流行,其中2014年埃博拉疫情在西非大暴发,给当地人民生活和全球公共健康带来极大挑战。埃博拉病毒能引起恶性出血热等症状,其致死率高,是迄今人类所知最致命的传染性病毒之一,并且临床上没有特效药物。

针对埃博拉病毒表面糖蛋白GP的抗体分离是抗病毒研究的一个重要方面,我们的合作者从埃博拉病毒感染康复病人体内分离到两株中和性抗体,mAb100和mAb114。这两种抗体不仅能在体外中和埃博拉病毒,对感染病毒后的动物也具有非常好的保护作用。

为阐明两种抗体的作用机制,本研究运用冷冻电子显微镜(Cryo-EM)和单颗粒三维重构(Single Particle 3D Reconstruction)的方法解析了GPdM-Fab100-Fab114三元复合物在pH 7.4和pH 5.0两种状态下的冷冻电镜结构,结合晶体学和生化实验结果,阐明两种抗体在病毒与宿主细胞膜融合过程的不同阶段发挥作用。

在入侵宿主细胞的过程中,埃博拉病毒首先会被宿主细胞摄入内吞体(Endosome)。病毒被呈递到晚期内吞体(Late Endosome)和溶酶体(Lysosome)的过程中,随着周围环境pH的降低,pH依赖的酶Cathepsin L(Cat L)和Cathepsin B(Cat B)会剪切GP,暴露出受体结合区域(Receptor binding domain, RBD)。GP受体结合区会与宿主细胞的受体蛋白NPC1相互作用,导致酶切后的GP发生构象变化,使得病毒的包膜与宿主细胞发生融合,并将病毒遗传物质释放到宿主细胞的细胞质中,实现病毒对宿主细胞的感染。

mAb100结合在GP底部,结合区跨越两个GP单体,并覆盖住GP的酶切位点(Protease cleavage site),在空间上阻碍了Cat L/B对GP蛋白的剪切。mAb114结合在GP头部的受体结合区域,能阻碍宿主细胞受体与GP的相互作用。两种抗体在中性pH和酸性pH下都能保持和GP的紧密结合,这使得其在内吞体和溶酶体等不同生理条件下一直起作用,通过阻碍GP蛋白介导的病毒膜融合的不同阶段,实现中和病毒的目的。

我们的研究表明,mAb100和mAb114能阻碍埃博拉病毒膜融合过程的薄弱环节,该过程为所有线状病毒科所共有,这对相关病毒的疫苗和药物开发具有很重要的指导作用。

英文摘要

Ebola virus(EBOV)is a negative-sense, enveloped single-strand RNA virus which belongs to the family Filoviridae. First discovered in African in 1976, EBOV has broken out for several times. During 2014-2015, the EBOV epidemic outbroke in West Africa and brought great challenges to people’s life as well as the global public health. EBOV causes a severe hemorrhagic fever with a high death rate. It is one of the world’s most fatal infectious virus and has no approved drugs yet.

Isolating antibodies targeting GP on the EBOV surface is an important way for the antiviral study. Our collaborations isolated two neutralizing antibodies, mAb100 and mAb114, from a human survival of the EBOV infection. These antibodies neutralize EBOV in vitro and can protect animals from EBOV infection as well.

To figure out the mechanism of these two antibodies, we determined the GPdM-fab100-fab114 ternary complex structures at pH 7.4 and pH 5.0 using Cryo-EM and single particle 3D reconstruction methods. Combined with the crystal and biochemical data, we illuminated that mAb100 and mAb114 function at different steps during the process of the EBOV membrane fusion with the host cell.

When EBOV entries the host cell, it is firstly uptaken into the endsome and is transfered to the late endosome and/or lysosome. As the pH decreases , pH dependent protease cathepsin L/B will cleave GP and expose the receptor binding domain(RBD). The RBD then interacts with the host cell receptor Niemann-Pick 1(NPC1), which triggers the cleaved GP to undergo conformational change. Then the virus membrane fuse with the host cell membrane and the virus genome is released to the host cell cytoplasma, implementing the virus infection of the host cell.

MAb100 binds at the base of GP, spans two GP promoters and covers the protease cleavage site, thus preventing the protease Cat L/B to cleave GP. MAb114 binds at the RBD and glycan cap of GP, which located at the head region, and hinders the host cell receptor to interact with the RBD. What’s more, mAb100 and mAb114 bind with GP at both neutral and acidic pH. This renders these two antibodies to function at physiological environment including the endosome and lysosome. By blocking different steps of the membrane fusion, mAb100 and mAb114 neutralize the EBOV.

To sum up, mAb100 and mAb114 target the vulnerabilities of the GP during membrane fusion, which are common among all members of the Filoviridae family. These study will be helpful to guide the vaccine and therapeutic development against the filovirus.

语种中文
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文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/125093
专题北京大学基础医学院
作者单位北京大学基础医学院
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桂淼. 埃博拉病毒表面糖蛋白GP与中和抗体复合物电镜结构研究[D]. 北京大学基础医学院. 北京大学,2016.
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