Investigating MHC-I downregulation by HIV-1 Nef using cryo-electron tomography with laser-based phase contrast

使用基于激光相差的冷冻电子断层扫描研究 HIV-1 Nef 对 MHC-I 的下调

• 批准号: 10607448
• 负责人: Petar Nikolov Petrov
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-08 至 2024-01-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10607448
• 关键词: Acquired Immunodeficiency Syndrome; Adaptor Signaling Protein; Antigen Presentation; Apoferritin; Benchmarking; Binding; Biology; Carbon; Cell surface; Cells; Clathrin; Clathrin Adaptor Protein Complexes; Clathrin Adaptors; Clathrin-Coated Vesicles; Communities; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Data; Development; Down-Regulation; Electron Microscope; Electrons; Elements; Film; Foundations; Frequencies; Generations; Goals; Guanosine Triphosphate Phosphohydrolases; HIV; HIV-1; Histocompatibility Antigens Class I; Human; Image; Immune; Immune response; Immune system; In Vitro; Lasers; Lysosomes; Major Histocompatibility Complex; Measures; Mediating; Membrane; Membrane Proteins; Methods; Modeling; Noise; Pathogenesis; Performance; Phase; Physiological; Play; Polymers; Proteins; Research; Resolution; Role; Route; Side; Signal Transduction; Specimen; Structure; T-Lymphocyte; Technology; Testing; Therapeutic; Thinness; Training; Transcription Factor AP-1; Validation; Viral; Viral Matrix Proteins; Virus; Visualization; Work; career; cofactor; electron optics; experimental study; human disease; imaging modality; insight; instrumentation; membrane reconstitution; new technology; particle; performance tests; protein transport; receptor; reconstitution; reconstruction; recruit; structural biology; therapeutic development; trafficking

Project Summary/Abstract Nef is a viral accessory protein which plays a critical role in the pathogenesis of HIV. Among other functions, it undermines the host immune response by downregulating MHC-I expression at the cell surface. By binding the clathrin adaptor AP-1 and its cofactor, the GTPase Arf1, Nef drives the formation of AP-1:MHC-I complexes. This sequesters MHC-I in clathrin-coated vesicles which are eventually trafficked to the lysosome. However, this mechanism is only partially understood. Cryo-electron tomography (cryo-ET) is a powerful method which can visualize the interactions between AP-1 and Nef on physiological membranes. Recent work in the Hurley lab used cryo-ET to visualize the interactions between Nef and AP-1 on membranes in vitro, finding that the complex forms a lattice. The reconstruction indicated that Nef stabilizes, but is not essential for, lattice formation, suggesting that an AP-1:Arf1 lattice forms as part of normal clathrin-mediated trafficking and that this lattice is hijacked by Nef to downregulate MHC-I. However, the resolution remains limited by the poor signal-to-noise ratio (SNR) of cryo-ET. In the proposed work, the SNR of cryo-ET will be increased in order to test this hypothesis with unprecedented resolution. To increase SNR, the laser phase plate (LPP), a technology developed in the Müller lab, will be integrated into a latest-generation aberration-corrected cryo-electron microscope. In Aim 1, the LPP will be benchmarked against standard, defocus-based cryo-electron microscopy by performing single- particle reconstructions of the purified model protein, apoferritin. This will demonstrate the first atomic resolution imaging with the LPP and thus introduce a powerful new imaging modality to the structural biology toolbox. In Aim 2, the work of the Hurley lab will be built upon by using the new imaging method of laser-based phase contrast cryo-ET to solve the structures of membrane-bound AP-1:Arf1 complexes in both the presence and absence of Nef. Comparison of the observed structures will elucidate the mechanism by which AP-1 assemblies are co-opted by Nef to downregulate MHC-I. In addition, these reconstructions will provide the first structural data of AP-1:Arf1 complexes on membranes and may reveal the structural basis for AP-1 and Arf1 residues associated with other human disease. More broadly, the proposed research will introduce a new technology, laser-based phase contrast cryo-ET, to the structural biology community. This work will also provide deep structural insights into trafficking in healthy and HIV-infected cells, which may be instrumental in uncovering the mechanisms of HIV pathogenesis and inform therapeutic approaches. This research will leverage the expertise of the Hurley lab in membrane reconstitution and HIV biology as well as the unique electron-optical instrumentation developed by the Müller lab. The training plan proposed in this application and the results of the project will serve as the foundation for a transition into an independent research career.

项目摘要/摘要 NEF是一种病毒辅助蛋白，在HIV的发病机理中起关键作用。在其他功能中， 它通过下调细胞表面的MHC-I表达来破坏宿主免疫反应。通过结合 网格蛋白适配器AP-1及其辅因子GTPase ARF1 Nef驱动AP-1：MHC-I配合物的形成。 这将在涂有网状蛋白涂层的蔬菜中隔离MHC-1，有时会被运送到溶酶体。但是，这个 机制仅部分理解。冷冻电子层析成像（Cryo-ET）是一种强大的方法，可以 可视化物理膜上AP-1和NEF之间的相互作用。赫利实验室的最新工作 使用Cryo-Et可视化体外膜上NEF和AP-1之间的相互作用，发现复合物 形成一个格子。重建表明，NEF稳定但不是必需的晶格形成， 建议形成AP-1：ARF1晶格作为正常网格蛋白介导的贩运的一部分，并且该晶格是 被NEF劫持以下调MHC-I。但是，该分辨率仍然受信噪比差的限制 （Cryo-Et的SNR）。在拟议的工作中，为了检验该假设，将增加冷冻-ET的SNR 具有前所未有的解决方案。为了增加SNR，激光相板（LPP）是在该技术中开发的技术 MüllerLab将集成到最新一代的低畸变校正的冷冻电子显微镜中。在AIM 1中， LPP将通过执行单个基于标准的基于散焦的冷冻电子显微镜进行基准测试 纯化的模型蛋白质蛋白质蛋白的颗粒重建。这将证明第一个原子分辨率 使用LPP进行成像，从而向结构生物学工具箱引入强大的新成像方式。在 AIM 2，将使用基于激光的阶段的新成像方法建立Hurley Lab的工作 对比的冷冻-ET解决膜结合的AP-1的结构：在存在和 缺乏nef。观察到的结构的比较将阐明AP-1组件的机制 由NEF选择下调MHC-I。此外，这些重建将提供第一个结构 AP-1：ARF1复合物上的数据，可能揭示AP-1和ARF1残差的结构基础 与其他人类疾病有关。更广泛地，拟议的研究将引入一项新技术， 基于激光的相比冷冻-ET与结构生物学界。这项工作还将提供深刻的 对健康和艾滋病毒感染细胞进行贩运的结构见解，这可能有助于发现 HIV发病机理和信息治疗方法的机制。这项研究将利用专业知识 在膜重建和艾滋病毒生物学的赫利实验室以及独特的电子光学 由Müller实验室开发的仪器。本应用程序提出的培训计划以及 项目将成为过渡到独立研究职业的基础。