Structural studies of Togaviruses

描述（由申请人提供）：晶体学是主要的结构工具，首次开启了近原子分辨率的病毒结构研究。然而，带有脂质包膜的大型病毒在结构上变化太大，其表面参与晶格接触的比例太小，无法形成稳定的晶体。冷冻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”，晶体学在确定可结晶的病毒成分方面起着辅助作用。在有利的情况下，低温电子显微图像的单粒子平均现在接近几乎原子分辨率，但是，再一次，这项技术的局限性在越来越复杂的多形性组装的研究中变得明显。冷冻电子断层扫描现在已经成为确定单个病毒颗粒结构的宝贵工具，而不依赖于对称等效组分之间的平均或相同均质颗粒的可用性。然而，层析重建的分辨率不足以确定原子细节的结构。因此，该拨款申请的一部分用于从断层扫描中提取更多信息，特别是用于稍微多形性，结构上基本上未知的风疹病毒。甲病毒和风疹病毒因其基因序列和形态相似而构成托加病毒科。在甲病毒中，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。我们计划扩展我们先前对二十面体含脂甲病毒的研究，并解决多形性风疹病毒更困难的结构分析。具体来说，我们正计划利用冷冻电子显微镜将基孔肯雅病毒样颗粒的分辨率提高到接近原子的分辨率，部分目的是为了帮助与美国国立卫生研究院疫苗研究中心的加里·纳贝尔一起设计一种针对基孔肯雅病毒的疫苗。我们还计划通过电子断层图分析来确定风疹病毒的结构。我们计划使用抗体和肽抑制剂来阻断与宿主细胞的成熟、附着和融合，以研究病毒生命周期中遇到的结构中间体。我们正计划确定风疹病毒成分的剩余未知结构，并使用晶体学和电子显微镜来研究它们彼此之间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.