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

新兴和人畜共患 RNA 病毒进入细胞的分子和结构基础

• 批准号: MR/L009528/1
• 负责人: Thomas Bowden
• 金额: $ 145.81万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FL009528%2F1
• 关键词: Molecular; Structural; Basis; Cell; Entry

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.

新出现的人畜共患病毒跨越了从动物到人类的物种障碍，反之亦然。它们在人类细胞中的繁殖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显示的受体分子结合来靶向特定细胞类型的能力。尽管这些病毒对动物和人类都造成了严重的疾病，并对全球健康和经济构成威胁，但人们对它们如何感染宿主知之甚少，事实证明，这些信息对于开发针对流感和HIV-1等研究更好的病毒的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的结合。从这些组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。我将阐明新出现的单链病毒与宿主细胞附着和融合的分子机制。这将在完整病毒和负责病毒进入的个别结构糖蛋白的背景下解决。这项研究仅限于来自三个科的紧急病毒：副粘病毒科(例如非洲亨尼帕病毒)、阿雷纳病毒科(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；支持的目标列表在表1中提供)。有四个互补的目标：1.病毒附着糖蛋白的重组表达和纯化，模型非致病病毒的制备，以及针对这些蛋白糖蛋白的中和抗体(NAB)的获得/发展。对病毒糖蛋白单独以及与细胞受体和NAB的复合体进行结晶学分析，以提供融合前和融合后组装的原子描述，并说明病毒进入所需的形态变化。对完整的、非致病性的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及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。突变病毒基因组，以产生修饰的病毒，以补充病毒宿主细胞进入的预测决定因素。

项目成果

Native functionality and therapeutic targeting of arenaviral glycoproteins.

• DOI: 10.1016/j.coviro.2016.04.001
• 发表时间: 2016-06
• 期刊: Current opinion in virology
• 影响因子: 5.9
• 作者: Crispin M;Zeltina A;Zitzmann N;Bowden TA
• 通讯作者: Bowden TA

Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection.

• DOI: 10.1093/infdis/jiv565
• 发表时间: 2016-04-01
• 期刊: The Journal of infectious diseases
• 作者: Dowall SD;Callan J;Zeltina A;Al-Abdulla I;Strecker T;Fehling SK;Krähling V;Bosworth A;Rayner E;Taylor I;Charlton S;Landon J;Cameron I;Hewson R;Nasidi A;Bowden TA;Carroll MW
• 通讯作者: Carroll MW

Determination of N-linked Glycosylation in Viral Glycoproteins by Negative Ion Mass Spectrometry and Ion Mobility.

• DOI: 10.1007/978-1-4939-2874-3_7
• 发表时间: 2015
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Bitto D;Harvey DJ;Halldorsson S;Doores KJ;Pritchard LK;Huiskonen JT;Bowden TA;Crispin M
• 通讯作者: Crispin M

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

使用 Jsubtomo 进行电子冷冻断层扫描重建的病毒包膜糖蛋白尖峰的平均值

• DOI: 10.3791/51714-v
• 发表时间: 2014
• 期刊: Journal of Visualized Experiments
• 作者: Bowden T

Uukuniemi Phlebovirus assembly and secretion leave a functional imprint on the virion glycome.

• DOI: 10.1128/jvi.01662-14
• 发表时间: 2014-09-01
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Crispin M;Harvey DJ;Bitto D;Halldorsson S;Bonomelli C;Edgeworth M;Scrivens JH;Huiskonen JT;Bowden TA
• 通讯作者: Bowden TA