Study of arenavirus assembly

COVID-19大流行对公共卫生、全球经济和社会稳定造成毁灭性影响 在过去两年中，强调了对人畜共患病病原体进行基础研究的重要性。本申请 描述了啮齿动物传播的人类病原体淋巴细胞脉络丛脑膜炎的结构和功能研究 病毒（LCMV），布尼亚病毒目中沙粒病毒科的成员。像其他成员一样， 在LCMV家族中，LCMV具有由大（L）和小（S）区段组成的负义、双区段基因组。 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区段编码病毒核蛋白（NP）和糖蛋白（GP）的前体。 糖蛋白复合物（GPC），其随后裂解成稳定的信号肽（SSP）、GP 1和GP 2。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被共包装 与膜相关的Z蛋白，也与嵌入在膜包膜的GP相互作用。 尽管已经通过X射线晶体学或低温电子显微镜（cro electron microscope）解决了来自AV的单个蛋白质的结构， 利用冷冻电镜（cryoEM），这些蛋白质在病毒体中的结构组织和组装机制 NP和RNA进入核衣壳的机制知之甚少。我们假设NP与基因组相互作用， RNA片段和L-RdRp形成核衣壳，其被募集到GP修饰的膜补丁 通过Z进行病毒体的出芽。拟议的结构和功能研究旨在验证这一假设， LCMV病毒粒子和核衣壳的组装技术刚刚建立了我们的团队在合作研究 水泡性口炎病毒（VSV），另一种负义RNA病毒。特别是低温电子断层扫描 （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图像中，以建立病毒体的结构框架。 目的1：研究GP、Z、NP和L蛋白之间的相互作用。近原子分辨率， 一种新的亚粒子重建方法将被用于成像完全组装的核衣壳组成的NP 蛋白质和基因组RNA片段来定义蛋白质-RNA相互作用的原子细节。核衣壳 结构将用于指导子粒子重建工作流程，并由原位结构补充 病毒粒子的核衣壳（Aim 2）。在这两个目标中，将进行结构指导的功能研究，以测试 LCMV核衣壳与病毒粒子组装机制假说。结构-功能关系相关 对病毒RNA合成的影响也将被探索。总的来说，预期的结果将提供新的见解 病毒体组装和病毒RNA合成的机制，不仅对于LCMV，而且对于一般的沙粒病毒。 拟议的研究利用了结构生物学的尖端技术，并将产生新的知识 目前任何沙粒病毒都无法获得的病毒结构。因此，创新研究应使独特的 通过加速抗病毒剂和疫苗的发现来控制未来的AV爆发。

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.