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

项目摘要/摘要 NEF是一种病毒辅助蛋白，在HIV的致病过程中起着关键作用。在其他功能中， 它通过下调细胞表面MHC-I的表达来破坏宿主的免疫反应。通过装订 网状蛋白适配器AP-1及其辅因子GTP酶Arf1，Nef驱动AP-1：MHC-I复合体的形成。 这将MHC-I隔离在包被网状蛋白的囊泡中，这些囊泡最终被输送到溶酶体。不过，这个 人们对这一机制的了解还只有一部分。冷冻电子断层扫描(Cryo-et)是一种强有力的方法，它可以 可视化AP-1和Nef在生理膜上的相互作用。赫尔利实验室最近的工作 使用冷冻-ET技术显示Nef和AP-1在体外膜上的相互作用，发现该复合体 形成晶格。重构表明，Nef稳定，但不是晶格形成所必需的， 这表明AP-1：Arf1晶格是作为正常的网格蛋白介导的转运的一部分形成的，并且这个晶格是 被Nef劫持以降低MHC-I的水平。然而，分辨率仍然受到低信噪比的限制 (SNR)。在所提出的工作中，将提高低温对撞机的信噪比以验证这一假设 以前所未有的决心。为了提高信噪比，激光相位板(LPP)是一项在 米勒实验室，将集成到最新一代的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，通过执行单一的- 纯化模型蛋白Apoferritin的颗粒重建。这将演示第一个原子分辨率 使用LPP进行成像，从而为结构生物学工具箱引入了一种强大的新成像方式。在……里面 目标2，赫尔利实验室的工作将建立在使用基于激光相位的新成像方法的基础上 对比CRYO-ET方法研究膜结合AP-1：ArF_1络合物的结构 尼夫不在。对观察到的结构的比较将阐明AP-1组装的机制 被Nef增选来下调MHC-I。此外，这些重建将提供第一个结构 膜上AP-1：Arf1复合体的数据，并可能揭示AP-1和Arf1残基的结构基础 与其他人类疾病有关。更广泛地说，这项拟议的研究将引入一项新技术， 基于激光的相位对比度低温，到结构生物界。这项工作也将为我们提供更深入的 对健康和艾滋病毒感染细胞贩运的结构性洞察，这可能有助于揭示 艾滋病毒的致病机制和治疗方法。这项研究将利用这些专业知识 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方面的研究 由米勒实验室开发的仪器。本申请书所建议的培训计划及 该项目将作为过渡到独立研究职业的基础。

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.