CryoEM structural analysis of Lassa virus GPC

拉沙病毒 GPC 的 CryoEM 结构分析

• 批准号: 9332757
• 负责人: Jack H Nunberg
• 金额: $ 21.18万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-20 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9332757
• 关键词: Address; Arenaviridae; Arenavirus; Binding; Biochemical; Biotin; Categories; Cell membrane; Cells; Chemistry; Chimeric Proteins; Classification; Cleaved cell; Collaborations; Complex; Cryoelectron Microscopy; Detergents; Development; Elements; Endosomes; Environment; Exposure to; GP2 gene; GTPBP1 gene; Genetic; Glycoproteins; Goals; Human; Image; Infection; Insecta; Intervention; Knowledge; Lassa virus; Life; Lipids; Mediating; Membrane; Membrane Fusion; Methodology; Molecular Conformation; Peptide Signal Sequences; Pharmacology; Photoaffinity Labels; Population; Public Health; Research; Resolution; Rodent; Sampling; Series; Structure; Transmembrane Domain; Vaccines; Viral; Viral Fusion Proteins; Viral Hemorrhagic Fevers; Virion; Virus; biodefense; combat; conformational conversion; experience; inhibitor/antagonist; innovation; insight; microscopic imaging; nanodisk; novel; novel therapeutics; novel vaccines; particle; pathogen; protein structure; receptor; reconstruction; small molecule; small molecule inhibitor; tomography

Hemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Intervention strategies that target the arenavirus envelope glycoprotein complex (GPC) hold promise for combating these lethal infections. In contrast to other class I fusion proteins, GPC contains three subunits: GP1, GP2 and a unique stable signal peptide (SSP) that acts in conjunction with GP2 to sense acidic pH in the endosome and trigger the structural transitions leading to virus-cell membrane fusion. Small-molecule inhibitors target this interaction to block arenavirus entry. A detailed understanding of GPC membrane fusion and its inhibition has been hampered by the lack of structural knowledge of the membrane-anchored GPC trimer. Crystallographic structures of soluble ectodomain fragments of GPC lack SSP and the transmembrane domain of GP2. We have determined a low-resolution structure of the Lassa virus (LASV) GPC on virion particles using cryo- electron microscopy (cryoEM) tomography, but were unable to resolve these missing elements. Remarkable technical advances in single-particle cryoEM now allow the capture of significantly more information than cryotomography and should enable us to generate high-resolution structures. In preliminary studies, we have produced LASV GPC in insect cells and used cryoEM to visualize the trimeric complex, in solution and embedded in lipid nanodiscs (NDs). This proposal entails a collaboration between two experienced research groups with complementary strengths in the biochemical analysis of arenavirus membrane fusion and single- particle cryoEM structure determination, with the goal of obtaining near-atomic resolution structural knowledge of the native LASV GPC complex in a membrane environment. We will pursue the following specific aims: (1) Determine the cryoEM structure of the precursor form of LASV GPC stabilized in lipid nanodiscs. NDs offer an ordered lipid environment to stabilize the structure of membrane proteins beyond the limits of nonionic detergents. We have incorporated cleavage-defective (cd) LASV GPC into NDs and will determine the cryoEM structure of the trimeric precursor. (2) Determine the structure of the mature prefusion form of LASV GPC. Purified wild-type LASV GPC contains a mixture of cleaved and uncleaved molecules. We will take advantage of our recent finding that only the mature complex is susceptible to photoaffinity labeling by fusion inhibitors to selectively enrich for cleaved GPC NDs. (3) Determine the low-pH structures of LASV GPC bound to its LAMP1 co-receptor and in its postfusion state. LASV GP1 undergoes a conformational switch at moderate endosomal pH to bind LAMP1. We have produced soluble LAMP1 and will determine the cryoEM structure of LAMP1-bound GPC NDs. Further acidification will trigger the fusogenic reorganization of the complex. Collectively, the structures will provide an unprecedented view of GPC and the conformational transitions that promote arenavirus membrane fusion and its inhibition. This knowledge will guide the development of novel vaccines and therapeutics against arenaviral hemorrhagic fevers.

出血热沙粒病毒对公共卫生和生物防御构成重大威胁。干预 针对沙粒病毒包膜糖蛋白复合物（GPC）的策略有望对抗这些疾病， 致命感染。与其他I类融合蛋白相比，GPC含有三个亚基：GP1、GP2和一个亚基。 独特的稳定信号肽（SSP），与GP 2共同作用，感知内体中的酸性pH， 触发导致病毒-细胞膜融合的结构转变。小分子抑制剂靶向于此 阻止沙粒病毒进入。详细了解GPC膜融合及其抑制， 由于缺乏膜锚定GPC三聚体的结构知识，晶体学 GPC的可溶性胞外域片段的结构缺乏SSP和GP 2的跨膜结构域。我们 已经确定了一个低分辨率的结构，拉沙病毒（LASV）GPC的病毒粒子使用冷冻- 电子显微镜（cryoEM）断层扫描，但无法解决这些缺失的元素。显著 单粒子cryoEM的技术进步现在允许捕获比 冷冻断层扫描应该能让我们生成高分辨率的结构在初步研究中， 在昆虫细胞中产生LASV GPC，并使用cryoEM使溶液中的三聚体复合物可视化， 包埋在脂质纳米盘（ND）中。这项建议需要两个经验丰富的研究人员之间的合作 在沙粒病毒膜融合和单- 粒子cryoEM结构测定，目的是获得近原子分辨率的结构知识 的天然LASV GPC复合物在膜环境中。我们将努力实现以下具体目标： (1)确定在脂质纳米盘中稳定的LASV GPC的前体形式的cryoEM结构。 ND提供了有序的脂质环境，以稳定膜蛋白的结构，使其超过脂质的限制。 非离子洗涤剂我们已经将裂解缺陷（cd）LASV GPC纳入ND中，并将确定 三聚体前体的cryoEM结构(2)确定的结构成熟的预融合形式的 LASV GPC纯化的野生型LASV GPC含有裂解和未裂解分子的混合物。我们将 利用我们最近的发现，即只有成熟的复合物对光亲和标记敏感， 融合抑制剂以选择性富集裂解的GPC ND。(3)确定LASV的低pH结构 GPC与其LAMP 1共受体结合并处于其融合后状态。LASV GP1经历构象 在中等内体pH下转换以结合LAMP 1。我们已经产生了可溶性LAMP 1，并将确定 LAMP 1结合的GPC ND的cryoEM结构。进一步的酸化将引发 复杂的。总的来说，这些结构将提供GPC和构象的前所未有的观点。 促进沙粒病毒膜融合及其抑制的转变。这些知识将引导 针对沙粒病毒出血热的新型疫苗和治疗剂的开发。