Molecular Determinants of Retroviral Capsid Morphology using a Novel Imaging Approach

项目摘要 未成熟的病毒颗粒结构可以为病毒颗粒在细胞内组装的性质提供有价值的线索。在……里面 在这项提议中，我们试图通过获得 使用冷冻电子显微镜(Cryo-EM)获得高分辨率结构。从不成熟的笑话中重建 不同类型的病毒样颗粒的结构显示了不同类型的GAG晶格组织之间的明显相似之处 迄今为止研究过的病毒。对人类免疫缺陷的比较分析增强了结构性洞察力 关于衣壳(CA)羧基末端重要性的1型病毒(HIV-1)冷冻-EM重建 结构域(CTD)和间隔肽1(SP1)区域在分子间形成CA六聚体组装中的作用 整体点阵结构的接触。为了从根本上了解逆转录病毒的组装和 未成熟粒子的成熟度、原子分辨率图是直方病毒亚科其他成员所必需的。 在这里，我提出了一种策略，即冷冻电子层析制导的单粒子重建(CRYO-ET制导 SPR)，以实现未成熟逆转录病毒颗粒的原子分辨率重建图。最初的努力是 简化EM方法以利用每个成像的优势来解析未成熟的GAG晶格 加工技术结合成了一种新的方法。我将用这种方法来研究二型艾滋病毒(HIV-2) 和人类T细胞白血病病毒1型(HTLV-1)。HTLV-1有一个不成熟的GAG晶格，形成了唯一的 在其他逆转录病毒形态中没有观察到的扁平衣壳，例如HIV-1。我的中央 假设HTLV-1 Gag CA蛋白在未成熟的Gag晶格中介导独特的分子间联系 这使得六聚体Gag构象能够产生不存在于未成熟体内的扁平形态 HIV病毒颗粒。通过研究HIV-2和HTLV-1的CA结构，我希望产生生物物理和 结构信息，以提供病毒颗粒组装和GAG体系结构的基本方面。要扩大规模 这项研究的生物学意义--正品HIV-2和HTLV-1的形态特征 未成熟的病毒颗粒衣壳晶格将被分析，并与更有序的类似病毒的未成熟病毒进行比较 用于高分辨率工作的粒子。双衣壳的单颗粒和层析冷冻-电磁混合方法 晶格将提供对这种扁平的CA形态以及 GAG在形成未成熟衣壳结构中的作用。确定搞笑格子组织中的结构差异 在密切相关的人类中，逆转录病毒揭示了病毒颗粒组装的新细节，最终将 确定针对Gag衣壳晶格形成的抗逆转录病毒治疗的新药物靶点。

Project Summary Immature virus particle structure can provide valuable clues to the nature of virus particle assembly in cells. In this proposal, we sought to decipher key structural details of immature retroviral Gag assemblies by obtaining high-resolution structures using cryo-electron microscopy (cryo-EM). Reconstructions from the immature Gag structure of distinct types of virus-like particles reveals distinct similarities among Gag lattice organization of the viruses studied to date. Structural insights have enhanced from a comparative analysis human immunodeficiency virus type 1 (HIV-1) cryo-EM reconstructions regarding the importance of the capsid (CA) carboxy-terminal domain (CTD) and spacer peptide 1 (SP1) regions in forming hexameric assemblies of CA in the intermolecular contacts of the overall lattice structure. In order to gain a fundamental understanding of retrovirus assembly and maturation, atomic resolution maps of immature particles for other members of Orthoretrovirinae are required. Here, I propose a strategy, i.e., cryo-electron tomography guided single particle reconstruction (Cryo-ET guided SPR), to achieve atomic resolution reconstruction maps of immature retrovirus particles. Initial efforts at streamlining the EM methodology to resolve immature Gag lattices using the strengths from each imaging processing technique combined into a new approach. I will apply this methodology to study HIV type 2 (HIV-2) and human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 has an immature Gag lattice that forms a uniquely flattened capsid that is not observed in other retroviruses morphologies, such as with HIV-1. My central hypothesis is that HTLV-1 Gag CA protein mediates unique intermolecular contacts in the immature Gag lattice that allows hexameric Gag conformations to generate a flattened morphology that does not exist within immature HIV viral particles. By studying the CA structures of HIV-2 and HTLV-1, I expect to generate biophysical and structural information to provide fundamental aspects of virus particle assembly and Gag architecture. To expand the biological significance of this proposed research morphological aspects of authentic HIV-2 and HTLV-1 immature virus particle capsid lattices will be analyzed and compaired to the more ordered viral-like immature particles used for high-resolution efforts. A single particle and tomography cryo-EM hybrid approach of both capsid lattices will provide novel insights into this flattened CA morphology as well as the conformational flexibility of Gag in forming immature capsid structures. Identifying structural differences in the Gag lattice organization among closely related human retroviruses sheds new details on virus particle assembly that will ultimately identify novel drug targets for antiretroviral therapies targeting Gag capsid lattice formation.