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

基本信息

  • 批准号:
    10607448
  • 负责人:
  • 金额:
    $ 6.95万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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.
项目总结/摘要 Nef是一种病毒辅助蛋白,在HIV的发病机制中起着关键作用。除其他功能外, 它通过下调细胞表面的MHC-I表达破坏宿主免疫应答。通过结合 网格蛋白衔接子AP-1及其辅因子GT α Arf 1、Nef驱动AP-1:MHC-I复合物的形成。 这将MHC-I隔离在网格蛋白包被的囊泡中,这些囊泡最终被运输到溶酶体。但这 机制只是部分理解。冷冻电子断层扫描(cryo-ET)是一种功能强大的方法, 可视化生理膜上AP-1和Nef之间的相互作用。赫尔利实验室最近的工作 使用cryo-ET观察Nef和AP-1在体外膜上的相互作用,发现复合物 形成格子。重建表明Nef稳定,但不是必不可少的,晶格形成, 这表明AP-1:Arf 1晶格是正常网格蛋白介导的运输的一部分, 被Nef劫持来下调MHC-I然而,分辨率仍然受到信噪比差的限制 (SNR)冷冻ET在所提出的工作中,为了检验这一假设,将增加冷冻ET的SNR 前所未有的决心。为了提高信噪比,激光相位板(LPP),一种在美国开发的技术, Müller实验室,将被集成到最新一代像差校正冷冻电子显微镜。在目标1中, LPP将以标准的、基于散焦的冷冻电子显微镜为基准, 纯化的模型蛋白脱铁铁蛋白的颗粒重建。这将展示第一个原子分辨率 LPP成像,从而为结构生物学工具箱引入一种强大的新成像模式。在 目标二,利用激光位相成像新方法,在Hurley实验室工作的基础上进一步发展 对比cryo-ET,以解决存在和不存在时膜结合AP-1:Arf 1复合物的结构。 没有Nef。观察到的结构的比较将阐明AP-1组装的机制, 被Nef用来下调MHC-I此外,这些重建将提供第一个结构 数据的AP-1:Arf 1复合物的膜,并可能揭示AP-1和Arf 1残基的结构基础 与其他人类疾病相关。更广泛地说,拟议中的研究将引入一种新技术, 基于激光的相衬冷冻ET,结构生物学社区。这项工作还将提供深入的 对健康和艾滋病毒感染细胞贩运的结构性见解,这可能有助于揭示 艾滋病毒的发病机制,并告知治疗方法。这项研究将利用专业知识, 赫尔利实验室在膜重建和艾滋病毒生物学以及独特的电子光学 Müller实验室开发的仪器。本申请书所提出的培训计划及 该项目将作为过渡到独立研究生涯的基础。

项目成果

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