Study of arenavirus assembly

沙粒病毒组装研究

• 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
• 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

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产生的对公共卫生，全球经济和社会稳定的破坏性影响19 在过去的两年中，强调了基础研究对人畜共患病原体的重要性。此应用程序 描述对啮齿动物的人类病原体淋巴细胞绒毛膜炎的结构和功能研究 病毒（LCMV），Bunyavirales命令中的Arenaviridae家族成员。像其他成员一样 family，LCMV具有负面意义，双段基因组，由大（L）和小（S）段组成。 L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 糖蛋白络合物（GPC）随后裂解成稳定的信号肽（SSP），GP1和GP2。在病毒体中， 与L蛋白相关的NP涂层L和S段的核蛋白质通过相互作用结合 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的单个蛋白质结构已通过X射线晶体学或冷冻电子求解 显微镜（冷冻），这些蛋白质在病毒体和装配机理中的结构组织 NP和RNA进入核蛋白酶的含量很少。我们假设NP与基因组相互作用 RNA片段和L RDRP形成一个核蛋白酶，该核素被募集到GP装饰的膜贴片 通过z萌芽。提出的结构和功能研究旨在检验 LCMV Virion和nucleocapsid组装具有我们团队在协作研究中建立的技术 囊泡气孔病毒（VSV），另一种负性RNA病毒。具体而言，冷冻电子断层扫描 （冷冻）将用于重建分子分辨率和原子的LCMV病毒粒子的第一个3D模型 单个蛋白质的模型将安装到Virion断层图中，以建立 病毒体并揭示GP，Z，NP和L蛋白之间的分子相互作用（AIM 1）。近乎原子分辨率 新型的亚颗粒重建方法将用于图像由NP组成的完全组装的核素 蛋白质和基因组RNA段以原子细节定义蛋白质RNA相互作用。核包膜 结构将用于指导下粒子重建工作流程，并由原位结构补充 来自病毒体的核蛋白酶（AIM 2）。在这两个目标中，结构引导的功能研究都将进行测试 LCMV Nucleocapsid和Virion的组装机制的假设。结构功能关系相关 还将探讨病毒RNA合成。总体而言，预期的结果将为您提供新的见解 病毒体组装和病毒RNA合成的机制，不仅用于LCMV，而且对于舞台病毒而言。 拟议的研究利用结构生物学的尖端技术，将产生新的知识 目前，任何竞技病毒目前都无法使用病毒结构。因此，创新研究应使其独特 通过加速抗病毒剂和疫苗的发现来控制未来的AV爆发。