Study of arenavirus assembly

沙粒病毒组装研究

基本信息

批准号：
10668498
负责人：
MING LUO
金额：
$ 54.02万
依托单位：
GEORGIA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-20 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10668498
关键词：
Acceleration Animals Antiviral Agents Architecture Arenavirus Basic Science Binding Binding Proteins Bunyavirales COVID-19 pandemic Cell membrane Cells Collaborations Complement Complex Cryo-electron tomography Cryoelectron Microscopy Data Density Gradient Centrifugation Deposition Disease Outbreaks Family Future GP2 gene GTPBP1 gene Genetic Transcription Genomic Segment Glycoproteins Human Image In Situ Individual Joints Junin virus Knowledge Lassa virus Lymphocyte Function Lymphocytic choriomeningitis virus Measures Membrane Messenger RNA Methods Modeling Molecular Mutation Nucleocapsid Nucleoproteins Old World Arenaviruses Peptide Signal Sequences Pichinde virus Polymerase Proteins Public Health RNA RNA Sequences RNA Viruses RNA chemical synthesis RNA-Directed RNA Polymerase RNA-Protein Interaction Resolution Rodent Series Signal Transduction Structure Structure-Activity Relationship System Techniques Technology Testing Tomogram Ultracentrifugation Vaccines Vesicular stomatitis Indiana virus Viral Viral Hemorrhagic Fevers Viral Proteins Virion Virus Virus Assembly X-Ray Crystallography Zoonoses density design experimental study future pandemic genomic RNA human pathogen image reconstruction innovation insight member novel novel strategies pathogen plasma protein Z prototype recombinant virus reconstruction recruit social structural biology three dimensional structure three-dimensional modeling vaccine development viral RNA

项目摘要

The devastating impact on public health, global economy and social stability incurred by the COVID-19 pandemic in the last two years has highlighted the importance of basic research into zoonotic pathogens. This application describes structural and functional studies into the rodent-borne human pathogen lymphocytic choriomeningitis virus (LCMV), a member of the Arenaviridae family in the Bunyavirales order. Like other members of the same family, LCMV has a negative sense, bi-segmented genome consisting of a large (L) and a small (S) segment. The L segment encodes the RNA-dependent RNA polymerase (L RdRp) protein and the multi-functional matrix protein (Z). The S segment encodes the viral nucleoprotein (NP) and the glycoprotein (GP) precursor of the glycoprotein complex (GPC) that is later cleaved into a stable signal peptide (SSP), GP1, and GP2. In the virion, nucleocapsids of NP coated L and S segments associated with the L protein are copackaged through interactions with membrane-associated Z proteins, which also interact with GPs embedded in the membrane envelope. Although structures of individual proteins from AVs have been solved by x-ray crystallography or cryo electron microscopy (cryoEM), the architectural organization of these proteins in the virion and the assembly mechanism of NP and RNA into the nucleocapsid are poorly understood. We hypothesize that NP interacts with genomic RNA segments and L RdRp to form a nucleocapsid, which is recruited to GP-decorated membrane patches through Z for budding of virions. The proposed structural and functional studies aim to test this hypothesis of LCMV virion and nucleocapsid assembly with techniques just established by our team in the collaborative studies of vesicular stomatitis virus (VSV), another negative sense RNA virus. Specifically, cryo electron tomography (cryoET) will be used to reconstruct the first 3D model of the LCMV virion at molecular resolution and atomic models of individual proteins will be fitted into the virion tomogram to establish the architectural framework of the virion and to unveil molecular interactions among GP, Z, NP and L proteins (Aim 1). Near-atomic resolution with novel sub-particle reconstruction method will be used to image fully assembled nucleocapsids consisting of NP protein and genomic RNA segment to define the protein-RNA interactions at atomic details. The nucleocapsid structure will be used to guide sub-particle reconstruction workflow and be complemented by in situ structures of nucleocapsids from virions (Aim 2). In both Aims, structure-guided functional studies will be performed to test hypotheses of assembly mechanisms of LCMV nucleocapsid and virion. Structure-function relationship relevant to viral RNA synthesis will also be explored. Overall, the anticipated results will provide new insights into the mechanism of virion assembly and viral RNA synthesis, not only for LCMV but also for Arenaviruses in general. The proposed studies harness cutting-edge technologies in structural biology and will generate new knowledge of viral structures currently unavailable to any of Arenaviruses. As such, the innovative studies shall make unique contributions by accelerating discoveries of antiviral agents and vaccines to control future AV outbreaks.

COVID-19 大流行对公共卫生、全球经济和社会稳定造成的破坏性影响过去两年凸显了人畜共患病原体基础研究的重要性。这个应用程序描述了对啮齿动物传播的人类病原体淋巴细胞性脉络膜脑膜炎的结构和功能研究病毒（LCMV），属于布尼亚病毒目沙粒病毒科的成员。和其他成员一样 LCMV 家族具有负义双节段基因组，由大片段 (L) 和小片段 (S) 组成。 L 片段编码 RNA 依赖性 RNA 聚合酶 (L RdRp) 蛋白和多功能基质蛋白质（Z）。 S段编码病毒核蛋白（NP）和糖蛋白（GP）前体糖蛋白复合物 (GPC)，随后裂解为稳定的信号肽 (SSP)、GP1 和 GP2。在病毒体中，与 L 蛋白相关的 NP 包被的 L 和 S 片段的核衣壳通过相互作用共同包装与膜相关的 Z 蛋白，它也与嵌入膜包膜中的 GP 相互作用。尽管 AV 中单个蛋白质的结构已通过 X 射线晶体学或冷冻电子解析显微镜（冷冻电镜），病毒体中这些蛋白质的结构组织和组装机制目前对 NP 和 RNA 进入核衣壳的过程知之甚少。我们假设 NP 与基因组相互作用 RNA 片段和 L RdRp 形成核衣壳，核衣壳被招募到 GP 修饰的膜片上通过 Z 表示病毒粒子的出芽。拟议的结构和功能研究旨在检验这一假设使用我们团队在合作研究中刚刚建立的技术进行 LCMV 病毒粒子和核衣壳组装水泡性口炎病毒（VSV），另一种负义RNA病毒。具体来说，冷冻电子断层扫描 (cryoET) 将用于以分子分辨率和原子分辨率重建 LCMV 病毒体的第一个 3D 模型单个蛋白质的模型将被拟合到病毒体断层扫描中，以建立病毒体的结构框架病毒粒子并揭示 GP、Z、NP 和 L 蛋白之间的分子相互作用（目标 1）。近原子分辨率新型亚粒子重建方法将用于对由 NP 组成的完全组装的核衣壳进行成像蛋白质和基因组 RNA 片段，以在原子细节上定义蛋白质-RNA 相互作用。核衣壳结构将用于指导亚粒子重建工作流程，并由原位结构补充来自病毒体的核衣壳（目标 2）。在这两个目标中，都将进行结构引导的功能研究来测试 LCMV核衣壳和病毒颗粒组装机制的假设。结构-功能关系相关病毒RNA合成也将被探索。总体而言，预期结果将为我们提供新的见解病毒粒子组装和病毒 RNA 合成的机制，不仅适用于 LCMV，也适用于一般沙粒病毒。拟议的研究利用结构生物学的尖端技术并将产生新知识目前任何沙粒病毒都无法获得病毒结构。因此，创新研究应具有独特性通过加速抗病毒药物和疫苗的发现来控制未来 AV 的爆发做出贡献。