Molecular and Structural Basis of Cell Entry by Emerging and Zoonotic RNA Viruses

新出现的人畜共患病病毒跨越物种屏障，从动物传播到人类，反之亦然。它们在人类细胞中繁殖的能力取决于多种因素，最重要的是，它们通过与宿主细胞表面上展示的受体分子结合而靶向特定细胞类型的能力。尽管在动物和人类中引起的疾病的严重性以及这些病毒对全球健康和经济构成的威胁，但人们对它们如何感染宿主知之甚少，这些信息已被证明对于开发抗病毒药物或基于抗体的药物非常有价值。利用结构生物学、免疫学和细胞生物学的技术，我的目标是以原子细节展示这些病毒附着和感染人类细胞的机制。这将涉及对分离分子的高分辨率结构研究与对完整病毒原型的低分辨率分析相结合。从这些综合技术中获得的信息最终将有助于我们对抗这些病原体的能力。我将阐明单链病毒与宿主细胞粘附和融合的分子机制。这将在完整的病毒和负责病毒进入的单个结构糖蛋白的背景下得到解决。本研究仅限于来自三个科的新出现的病毒：副粘病毒科（例如非洲亨尼帕病毒）、沙粒病毒科（例如委内瑞拉出血热病毒）和布尼亚病毒科（例如汉坦病毒肺综合征相关疾病;靶标列表见支持案例，表1）。病毒附着糖蛋白的重组表达和纯化、模型非致病性病毒的制备以及针对这些糖蛋白的中和抗体（nAb）的获得/开发。对单独的病毒糖蛋白和与细胞受体和nAb复合的病毒糖蛋白进行晶体学分析，以提供融合前和融合后组装体的原子描述，并说明病毒进入所需的形态学变化。对完整的非致病性病毒同源物进行冷冻电子显微镜分析，以可视化这些病毒的超微结构，其亚单位糖蛋白的组装，并为整个病毒粒子的-受体和-抗体相互作用提供结构基础。病毒基因组的诱变，以产生经修饰的病毒，以补充病毒进入宿主细胞的结构预测决定簇。

Emerging zoonotic viruses cross the species barrier from animals to humans or vice-versa. Their ability to propagate in human cells is dependent on a variety of factors, most importantly, their ability to target particular cell types by binding to receptor molecules displayed on the surface of the host cell. Despite the severity of disease caused in both animals and humans and threat these viruses pose to global health and economy, little is known of how they infect their host, information which has proven to be extremely valuable for the development of antiviral drugs or antibody-based medicines for better-studied viruses such as flu and HIV-1. Using techniques in structural biology, immunology, and cell biology, I aim to show in atomic detail the mechanism by which these viruses attach to and infect human cells. This will involve the combination of high-resolution structural studies on isolated molecules with lower resolution analyses of intact viral prototypes. The information derived from these combined techniques will ultimately aid in our ability to combat these pathogens. I will elucidate the molecular mechanism by which by emergent single-stranded viruses undergo attachment and fusion with host cells. This will be addressed in the context of both intact virus and the individual structural glycoproteins responsible for viral entry. This investigation is limited to emergent viruses from three families: Paramyxoviridae (e.g. African Henipaviruses), Arenaviridae (e.g. Venezuelan Hemorrhagic fever virus), and Bunyaviridae (e.g. Hantavirus Pulmonary Syndrome associated diseases; a list of targets is provided in the Case for Support, Table 1).There are four complementary goals:1. Recombinant expression and purification of viral attachment glycoproteins, preparation of model non-pathogenic viruses, and the acquisition/development of neutralising antibodies (nAbs) against these glycoproteins.2. Crystallographic analysis of viral glycoproteins alone and in complex with cellular receptors and nAbs to provide atomic descriptions in pre- and post-fusion assemblies, and illustrate the morphological changes required for viral entry.3. Cryo-electron microscopic analysis of intact, non-pathogenic viral orthologues to visualise the ultrastructure of these viruses, the assembly of their subunit glycoproteins, and to provide a structural basis for -receptor and -antibody interactions for entire virions.4. Mutagenesis of the viral genome to generate modified viruses to complement structure predicted determinants for virus host cell entry.