Deciphering the Molecular Assembly Mechanism of Giant DNA Viruses

项目摘要/摘要 在过去的二十年里，已经发现了许多巨大的DNA病毒，其中一些比 小牢房。这些巨大的病毒是如何用数千个简单的蛋白质构建块组装它们的病毒粒子外壳的 确切地说，这是一个谜。然而，这些病毒的巨大规模对目前构成了重大挑战 可用的技术。这个项目将通过使用海洋巨型病毒自助餐厅来应对这一挑战 Roenbergensis病毒(CroV)作为破译巨型病毒组装机制的模型，以及作为一种 有机会开发技术，将这些巨大结构的分辨率极限推向原子水平。 在过去的五年中，冷冻电子显微镜(Cryo-EM)已经成为一种日益强大的工具 在原子分辨率下研究生物分子结构，为其开发者赢得了2017年诺贝尔奖 化学。我们将使用配备了最新新技术的最先进的低温EM收集更高质量的图像 硬件，如能量过滤器、直接电子探测器和相位板。将这些图像与 新的软件算法，我们将通过图像分析来确定巨型CroV的结构以达到高分辨率 和重建。单个CroV蛋白的结构也将通过Cryo-EM解析到原子分辨率 使用各种方法并对接成低温EM重建的地图。由此产生的伪原子 结构将允许表征CroV的超微结构特征和结构，构建 解开巨型病毒组装的必要基础。结构信息将与 经典的生物物理、分子动力学模拟、数学建模和计算分析 评估新的巨型病毒组装模型。在新的组装模型中，巨人的蛋白质壳 病毒以有趣的螺旋方式从5个顶点连续组装而不是组装 以先前假设的循序渐进的方式从补丁开始。巨型病毒蛋白外壳由蛋白质组装而成 类似于其他病毒的构建块，包括许多人类病原体。一些巨大的病毒已经被 与肺炎和认知功能改变等人类疾病有关。了解这些 控制巨型病毒组装的原则将促进治疗药物的开发 病毒组装，从而为一般预防和治疗病毒疾病提供了一条新的途径。澄清 驱动这些巨型病毒组装的分子相互作用也将揭示如何控制 有效的蛋白质-蛋白质相互作用，有助于合理设计大尺寸的病毒样纳米颗粒 用于生物医学和其他纳米应用的范围。由于一些巨大的病毒比一个小细胞还大， 该项目开发的技术和方法将推动结构生物学的极限，并提供新的 有用的工具来研究更大的超分子组装，并最终在未来研究整个细胞。

Project Summary/Abstract Over the last two decades, many giant DNA viruses have been discovered, some of which are bigger than a small cell. How these giant viruses assemble their virion shell from thousands of simple protein building blocks so precisely is a mystery. However, the sheer size of these viruses poses a significant challenge to currently available techniques. This project will tackle this challenge by using a marine giant virus Cafeteria roenbergensis virus (CroV) as a model to decipher the assembly mechanism of giant viruses, and as an opportunity to develop technology to push the resolution limit of these gigantic structures to the atomic level. During the last five years, cryo-electron microscopy (cryo-EM) has become an increasingly powerful tool to study the structures of biological molecules at atomic resolution, earning its developers the 2017 Nobel Prize in Chemistry. We will collect higher quality images using state-of-the-art cryo-EM equipped with latest new hardware, such as energy filters, direct electron detectors and phase plates. Using these images together with new software algorithms, we will determine the structure of giant CroV to high resolution by image analyses and reconstruction. Structures of individual CroV proteins will also be solved to atomic resolution by cryo-EM using various methods and docked into the cryo-EM reconstructed maps. The resultant pseudo-atomic structure will allow characterization of the ultrastructural features and architecture of CroV, building the essential foundation to unravel the assembly of giant viruses. The structure information will be combined with classic biophysical, molecular dynamic simulation, mathematical modeling, and computational analyses to evaluate the novel assembly model of giant viruses. In the new assembly model, the protein shells of giant viruses are assembled continuously from the 5-fold vertices in an interesting spiral way instead of assembled from patches in a step-wise fashion previously assumed. Giant virus protein shell is assembled from protein building block similar to other viruses, including many human pathogens. Some giant viruses have been associated with human diseases such as pneumonia and cognitive functional change. Understanding these principles governing the assembly of giant viruses will improve the development of therapeutic agents to inhibit virus assembly, thus providing a new avenue for preventing and treating viral diseases in general. Elucidation of the molecular interactions that drive assembly of these giant viruses will also shed light on how to control protein-protein interactions effectively, facilitating the rational design of virus-like nanoparticles with a wide size range for biomedical and other nano-applications. Since some giant viruses are bigger than a small cell, techniques and methods developed in this project will push the limits of structural biology and provide new and useful tools to study even larger supramolecular assemblies and eventually the whole cell in the future.