Study of arenavirus assembly

新冠肺炎疫情对公众健康、全球经济和社会稳定造成的破坏性影响 在过去的两年中，强调了对人畜共患病病原体进行基础研究的重要性。此应用程序 描述了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Arenaviridae家族的成员。像同一组织的其他成员一样 与烟草花叶病毒属同源，具有负义，基因组由大片段(L)和小片段(S)组成。 L片段编码依赖核糖核糖核酸聚合酶(L RdRp)蛋白和多功能基质 蛋白质(Z)。S片段编码病毒核蛋白(NP)和糖蛋白(GP)前体。 糖蛋白复合体(GPC)，后来被裂解成稳定信号肽(SSP)、GP1和GP2。在病毒粒子中， 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而被套装 与膜相关的Z蛋白，它也与嵌入在膜被膜中的GP相互作用。 虽然aVs中单个蛋白质的结构已经通过x射线结晶学或冷冻电子进行了解析 显微镜(冷冻EM)，这些蛋白质在病毒粒子中的结构组织和组装机制 NP和RNA进入核衣壳的情况知之甚少。我们假设NP与基因组相互作用 RNA片段与L RdRp形成核衣壳，被招募到GP修饰的膜片上 通过Z表示病毒粒子的萌发。拟议的结构和功能研究旨在检验这一假说 LCMV病毒粒子和核衣壳组装技术 水疱性口炎病毒(VSV)是另一种负义RNA病毒。具体地说，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病毒粒子的第一个3D模型 单个蛋白质的模型将被安装到病毒粒子断层图中，以建立 并揭示GP、Z、NP和L蛋白之间的分子相互作用(目标1)。具有近原子分辨率 新的亚粒子重建方法将用于成像由NP组成的完全组装的核衣壳 蛋白质和基因组RNA片段，在原子细节上定义蛋白质-RNA相互作用。核衣壳 将使用结构来指导子粒子重建工作流程，并以原位结构为补充 病毒粒子中的核衣壳(目标2)。在这两个目标中，将进行结构指导的功能研究来测试 LCMV核衣壳和病毒粒子组装机制的假说。结构-功能关系相关 对病毒RNA的合成也将进行探索。总体而言，预期结果将为 病毒粒子组装和病毒RNA合成的机制，不仅对LCMV，而且对一般的Arena病毒也是如此。 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 目前任何一种阿拉伯病毒都无法获得的病毒结构。因此，创新研究将使其独树一帜 通过加速发现抗病毒药物和疫苗来控制未来的病毒爆发，从而做出贡献。

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.