Partial maturation in mosquito-borne flaviviruses: developing new approaches to characterize the role of lattice heterogeneity in fusion, infectivity, and antibody neutralization

项目摘要 当蚊子传播的黄病毒遇到细胞时，它必须粘附在细胞表面的受体上，内吞， 最终与内体膜融合。为了实现这一点，黄病毒使用180 E的外部晶格 蛋白质在中性pH值下排列成90个反平行二聚体的人字形排列。 内体诱导广泛的构象重排，导致60个向外的三聚体E尖峰 它可以嵌入宿主细胞膜并将细胞膜拉到一起，使它们融合并释放病毒 RNA进入宿主细胞质。尽管黄病毒格被规范地描述为90个平放的二聚体（成熟的 状态）或60个三聚体朝外（不成熟和融合状态），高达50%的登革病毒体具有不完全的 成熟导致具有不同取向的E二聚体和三聚体的镶嵌图案。尚不清楚 镶嵌晶格是否由于空间位阻而比它们的完美对应物功能性差， 构象重排，或者可能有某些优势，解释了为什么进化 保留了它们的异质排列。这对治疗方法的设计有很大的影响， 成熟和未成熟的斑块具有不同的表位暴露和可能不同的结合亲和力， 这些抗体还不太清楚。 我们将通过对马赛克的结构和功能研究来探讨这个问题 登革热和西尼罗河病毒，使用冷冻电子断层扫描和亚断层扫描平均，以确定 在病毒晶格中单个E蛋白的位置和方向，并设计新的分析来描述 病毒群体的异质性。我们将评估镶嵌表面如何影响融合和抗体 结合，并通过成像病毒与靶标相互作用直接可视化结构-功能关系 膜和细胞。这种方法将使我们能够识别马赛克晶格的哪些区域参与 粘附或融合，以及功能性病毒粒子是否有利于更异质或均匀的表面。电流 结构生物学通常在含有大量非活性病毒体的样品上进行;通过成像 直接功能状态，从分析中消除无活性的病毒体，以便于鉴定结构 在基于结构的治疗和疫苗设计中应优先考虑的病毒状态。 虽然这项工作将集中在黄病毒格，功能格是无处不在的所有领域的生活， 在人类健康和疾病中发挥不可或缺的作用。我们开发的研究黄病毒的方法和分析将 直接适用于其他病毒，但也可能有助于理解细菌等不同的过程 趋化性、碳固定和人类心肌收缩。

PROJECT SUMMARY When a mosquito-borne flavivirus encounters a cell, it must adhere to a receptor on the cell surface, endocytose, and finally fuse with the endosomal membrane. To accomplish this, flaviviruses use an exterior lattice of 180 E proteins arrayed in a herringbone arrangement of 90 antiparallel dimers at neutral pH. Acidification in the late endosome induces a widespread conformational rearrangement, resulting in 60 outward-facing trimeric E spikes that can embed into the host membrane and pull the membranes together, fusing them and releasing the viral RNA into the host cytoplasm. Although flavivirus lattices are canonically described as 90 dimers lying flat (mature state) or 60 trimers facing outward (immature and fusion states), up to 50% of Dengue virions have incomplete maturation that results in a mosaic pattern of E dimers and trimers with different orientations. It is unknown whether mosaic lattices are less functional than their perfect counterparts due to steric hindrance of the conformational rearrangements, or alternatively might have certain advantages that explain why evolution has conserved their heterogeneous arrangement. This has strong consequences for the design of therapeutics, as the mature and immature patches have different epitope exposure and possibly different binding affinities for antibodies that are not well understood. We will explore this question through a combination of structural and functional studies performed on mosaic Dengue and West Nile viruses, using cryo-electron tomography and subtomogram averaging to determine the position and orientation of individual E proteins within the viral lattices, and designing new analyses to describe the heterogeneity of the viral population. We will evaluate how the mosaic surface affects fusion and antibody binding, and directly visualize the structure-function relationship by imaging viruses interacting with target membranes and cells. This approach will allow us to identify which areas of a mosaic lattice participate in adhesion or fusion, and whether functional virions favor more heterogeneous or homogeneous surfaces. Current structural biology is usually performed on samples that contain a large number of inactive virions; by imaging functional states directly, inactive virions are eliminated from analysis to facilitate identification of the structural states of the virus that should be prioritized in structure-based therapeutic and vaccine design. While this work will focus on the flavivirus lattice, functional lattices are ubiquitous in all the domains of life and play integral roles in human health and disease. The methods and analyses we develop to study flaviviruses will directly apply to other viruses, but will also potentially aid in understanding processes as diverse as bacterial chemotaxis, carbon fixation, and human cardiac muscle contraction.