Molecular Determinants of Retroviral Capsid Morphology using a Novel Imaging Approach

使用新型成像方法研究逆转录病毒衣壳形态的分子决定因素

• 批准号: 10012546
• 负责人: Nathaniel Lee Talledge
• 金额: $ 6.98万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-17 至 2021-09-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10012546
• 关键词: 3-Dimensional; Alpha Particles; Anti-Retroviral Agents; Architecture; Benchmarking; Biogenesis; Biological; Biophysics; Capsid; Capsid Proteins; Cell membrane; Cells; Complex; Core Protein; Crowding; Cryo-electron tomography; Cryoelectron Microscopy; Disease; Drug Design; Drug Targeting; Electron Microscopy; Elements; Foundations; Goals; HIV; HIV-1; HIV-2; Health; Human; Human T-lymphotropic virus 1; Hybrids; Image; Individual; Knowledge; Life Cycle Stages; Location; Maps; Mediating; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Morphology; Nature; Outcome; Pathway interactions; Peptides; Pharmacotherapy; Positioning Attribute; Process; Production; Property; Proteins; Radiation induced damage; Research; Resolution; Retroviridae; Sampling; Structure; Techniques; Testing; Tomogram; Tube; Ursidae Family; Viral; Virion; Virus; Virus Assembly; Virus Diseases; Virus-like particle; antiretroviral therapy; base; comparative; drug development; electron radiation; flexibility; gag Gene Products; image processing; imaging approach; insight; member; new therapeutic target; novel; novel strategies; particle; reconstruction; targeted treatment; tool

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.

项目摘要 未成熟的病毒颗粒结构可以为病毒颗粒在细胞中组装的性质提供有价值的线索。在 根据这项提议，我们试图通过获得以下信息来破译未成熟逆转录病毒Gag组装体的关键结构细节： 使用冷冻电子显微镜（cryo-EM）的高分辨率结构。从未成熟的加格重建 不同类型的病毒样颗粒的结构揭示了不同类型病毒的Gag晶格组织之间的明显相似性。 迄今为止研究过的病毒。从比较分析人类免疫缺陷综合征 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 整个晶格结构的接触。为了获得对逆转录病毒组装的基本了解， 为了使病毒成熟，需要正逆转录病毒亚科其他成员的未成熟颗粒的原子分辨率图。 在这里，我提出了一个策略，即，冷冻电子断层扫描引导的单粒子重建（冷冻-ET引导的 SPR），以实现未成熟逆转录病毒颗粒的原子分辨率重建图。初步努力， 简化EM方法，以使用每个成像的优势来解析未成熟的Gag晶格 加工技术结合成一种新的方法。我将应用这种方法来研究艾滋病毒2型（HIV-2） 和人T细胞白血病病毒1型（HTLV-1）。HTLV-1有一个不成熟的Gag晶格， 在其他逆转录病毒形态中未观察到的扁平衣壳，例如HIV-1。我的中枢 假设HTLV-1 Gag CA蛋白介导未成熟Gag晶格中独特的分子间接触 这使得六聚体Gag构象能够产生不存在于未成熟Gag构象中的扁平形态， HIV病毒颗粒。通过研究HIV-2和HTLV-1的CA结构，我期望产生生物物理和生物化学的结果。 结构信息，以提供病毒颗粒组装和Gag结构的基本方面。扩大 这项研究的生物学意义，真正的HIV-2和HTLV-1的形态学方面 不成熟的病毒颗粒衣壳晶格将被分析并与更有序的病毒样不成熟的病毒颗粒衣壳晶格进行比较。 用于高分辨率工作的粒子。两种衣壳的单粒子和层析成像cryo-EM混合方法 晶格将提供新的见解，这种扁平CA形态以及构象的灵活性， 在形成未成熟的衣壳结构时受阻。识别Gag晶格组织中的结构差异 在密切相关的人类逆转录病毒中揭示了病毒颗粒组装的新细节， 鉴定用于靶向Gag衣壳晶格形成的抗逆转录病毒疗法的新药物靶标。