Structural studies of Togaviruses

描述(申请人提供：结晶学是首次开启近原子分辨率的病毒结构研究的主要结构工具。然而，具有脂质包膜的较大病毒的结构变化太大，其表面参与晶格接触的比例太小，无法形成稳定的晶体。低温电子显微镜后来成为推进结构生物学的主要工具，给出了“大图景”，结晶学在确定可结晶的病毒成分方面发挥了辅助作用。在有利的情况下，冷冻电子显微镜图像的单粒子平均现在接近几乎原子的分辨率，但在研究日益复杂的多形组件时，这项技术的局限性再次变得明显起来。冷冻电子断层扫描现在已经成为一种非常有价值的工具，用于确定单个病毒颗粒的结构，而不需要依赖对称等价成分之间的平均或相同的均匀颗粒的可用性。然而，断层重建的分辨率不足以确定原子细节的结构。因此，这项赠款申请的一部分专门用于从断层图像中提取更多信息，特别是应用于稍具多形性、结构上基本上未知的风疹病毒。甲型病毒和风疹病毒由于在基因序列和形态上的相似性而构成TogaVirus家族。在甲型病毒中，基孔肯雅病毒是一个主要的世界卫生问题，因为它最近在亚洲和非洲重新出现。我们计划扩展我们之前对二十面体、含脂甲型病毒的研究，并解决更困难的多形性风疹病毒的结构分析。具体地说，我们计划使用低温电子显微镜将基孔肯雅病毒样颗粒的分辨率扩展到近原子分辨率，这在一定程度上是为了帮助与美国国立卫生研究院疫苗研究中心的加里·纳贝尔一起设计针对基孔肯雅病毒的疫苗。我们还计划通过电子断层图像分析来确定风疹病毒的结构。我们计划使用抗体和多肽抑制剂来阻止成熟、附着和与宿主细胞的融合，以研究病毒生命周期中遇到的结构中间体。我们计划确定风疹病毒成分的剩余未知结构，并使用结晶学和电子显微镜来研究它们之间的相互作用以及与抗体的相互作用。

DESCRIPTION (provided by applicant: Crystallography was the primary structural tool that first opened up structural investigation of viruses at near-atomic resolution. However, larger viruses with lipid envelopes are too variable in their structures and have too small a proportion of their surface involved in lattice contacts to make stable crystals. Cryo-electron microscopy later emerged as the leading tool for advancing Structural Biology to give the "big picture", with crystallography playing a supporting role in the determination of the crystallizable viral components. Single particle averaging of cryo-electron microscopic images is now approaching almost atomic resolution in favorable cases but, once again, the limits of this technology are becoming apparent in the investigations of progressively more complex pleomorphic assemblies. Cryo-electron tomography has now emerged as an invaluable tool for the determination of the structure of individual viral particles without reliance on averaging between symmetrically equivalent components or on the availability of identical homogeneous particles. However, the resolution of a tomographic reconstruction is insufficient to determine structure in atomic detail. Thus, a part of this grant application is dedicated towards extracting more information from tomograms, especially as applied to the slightly pleomorphic, structurally largely unknown, Rubella virus. Alphaviruses and Rubella virus constitute the Togavirus family because of their similarity in gene order and morphology. Among alphaviruses, Chikungunya virus is a major world health concern because of its recent re-emergence in Asia and Africa. We plan to extend our previous studies of the icosahedral, lipid containing alphaviruses, and tackle the more difficult structural analysis of pleomorphic Rubella virus. Specifically, we are planning to extend the resolution of Chikungunya virus-like particles to near-atomic resolution using cryo-electron microscopy in part as an aid to the design of a vaccine against Chikungunya virus with Gary Nabel at the NIH Vaccine Research Center. We also plan to determine the structure of Rubella virus by analyses of electron tomograms. We plan to use antibodies and peptide inhibitors to block maturation, attachment and fusion with host cells in order to study the structural intermediates encountered in the viral life cycle. We are planning to determine the remaining unknown structures of the Rubella virus components and use both crystallography and electron microscopy to study their interactions with each other and with antibodies.