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

基本信息

批准号：
10219047
负责人：
Nathaniel Lee Talledge
金额：
$ 6.88万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-17 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10219047
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

项目摘要

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细胞中病毒颗粒组装的性质提供宝贵的线索。在这项建议，我们试图通过获得未成熟逆转录病毒插科打术的关键结构细节。使用冷冻电子显微镜（Cryo-EM）的高分辨率结构。未成熟插科打的重建不同类型的类似病毒的颗粒的结构揭示了插科打晶格组织之间的独特相似之处迄今为止研究的病毒。比较分析人类免疫缺陷的结构见解增强了病毒类型1（HIV-1）冻干的重建有关CAPSID（CA）羧基末端的重要性域（CTD）和间隔肽1（SP1）区域，在分子间形成Ca的六聚体组件中整体晶格结构的接触。为了获得对逆转录病毒大会的基本了解，需要针对异源性静脉曲素的其他成员的未成熟颗粒的成熟，原子分辨率图。在这里，我提出了一种策略，即低温电子断层扫描引导的单个粒子重建（Cryo-Et指导 SPR），以实现未成熟逆转录病毒颗粒的原子分辨率重建图。最初的努力使用每种成像中的强度来简化EM方法来解决未成熟的堵塞晶格处理技术合并为一种新方法。我将使用这种方法来研究HIV 2型（HIV-2）和人类T细胞白血病病毒1型（HTLV-1）。 HTLV-1具有未成熟的插科打晶格，形成了独特的在其他逆转录病毒形态（例如HIV-1）中未观察到的扁平衣壳。我的中央假设是HTLV-1 GAG CA蛋白介导了未成熟的GAG晶格中独特的分子间接触这使得六聚体的插入构象可以产生一种不成熟内不存在的扁平形态 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可提供病毒颗粒组装和堵嘴结构的基本方面。扩展这项提出的研究形态学方面的生物学意义是真实的HIV-2和HTLV-1 未成熟的病毒颗粒衣壳晶格将进行分析，并与更有序的病毒样不成熟用于高分辨率工作的颗粒。两种衣壳的单个粒子和层析成像冷冻杂种方法格子将为这种扁平的CA形态以及构象的灵活性提供新颖的见解形成未成熟的衣壳结构时的插科打。确定插科打晶格组织的结构差异在密切相关的人类逆转录病毒中，提供了有关病毒颗粒组件的新细节，该细节最终将确定针对靶向og胶状晶格形成的抗逆转录病毒疗法的新型药物靶标。