Structure Analysis of Viral Assembly Mechanisms

病毒组装机制的结构分析

基本信息

批准号：
10179316
负责人：
Mark Jay Yeager
金额：
$ 59.98万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2022-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10179316
关键词：
3-Dimensional AIDS/HIV problem Acquired Immunodeficiency Syndrome Address Affect Architecture Binding Biological C-terminal Capsid Cell Nucleus Chemicals Chicago Complex Computer Models Computer Simulation Cone Cryoelectron Microscopy Crystallization Crystallography Data DegP protease Disulfides Docking Drug resistance Electrostatics Fissural Freezing Fullerenes Generations Goals Grain HIV-1 HIV-1 protease Hydration status Hydrophobic Interactions Image Analysis Label Length Ligation Maps Medical Methods Micro Electron Diffraction Modeling Molecular Molecular Conformation Mutate N-terminal NMR Spectroscopy Nuclear Pore Pathway interactions Peptide Hydrolases Peptides Pharmaceutical Preparations Productivity Protein Biosynthesis Proteins Protomer Recombinants Research Research Personnel Resolution Roentgen Rays S Phase SP1 gene Site Solid Structure Techniques Testing Thinness Time United States National Institutes of Health Utah Viral Virus Virus Assembly X-Ray Crystallography atomic data biophysical techniques crosslink crystallinity design drug action drug discovery electron crystallography experimental study inhibitor/antagonist insight molecular dynamics multi-scale modeling mutant novel novel therapeutic intervention particle programs reconstruction resistance mechanism self assembly simulation solid state nuclear magnetic resonance success therapeutic target therapeutically effective three dimensional structure

项目摘要

ABSTRACT A theme of our research program is the design and assembly of symmetric surrogates of the quasi- hexagonal and pleiomorphic lattices of the HIV-1 immature and mature capsids that enable high-resolution structure analysis by cryoEM, microED, X-ray crystallography and ssNMR spectroscopy. Interpretation of our experimental results is fortified by the addition of multi-scale, computational simulations. · Aim 1: Structural studies of the immature Gag lattice: We previously determined a 3.2 Å X-ray structure of a CTD-SP1 Gag construct, which revealed a 6-helix bundle comprised of 2 turns of CTD and 2 turns of SP1. The protease cleavage sites are sequestered in the interior of the bundle. This surprising result revealed a mystery: How does protease gain access to the sequestered cleavage sites? ssNMR spectroscopy of selectively labeled ΔMA-Gag VLPs will allow us to examine conformational dynamics of the junction helices. Time-resolved cryoEM of 2D crystals of CTD-SP1, which diffract to 5-Å resolution, will test whether protease cleavage is initiated at the fissures within the immature Gag lattice. MicroED of 2-4 µm 3D crystals yielded a 2.9-Å resolution 3D structure of CTD-SP1 with bound bevirimat, which sets the stage for us to explore mechanisms of drug resistance and action of second-generation maturation inhibitors. Our results will not only provide data for atomic-resolution MD simulations, but also help develop and refine novel coarse-grained (CG) models of Gag lattice components to explore the dynamics of on- and off-pathway generation of viral particles. · Aim 2: Structural studies of the mature capsid: We previously used disulfide crosslinking to stabilize and solve X-ray structures of the CA hexamer and pentamer. The resulting atomic model of the fullerene cone suggested mechanisms for the continuously varying curvature in the conical capsid. We also showed that the compound PF74, a CPSF6 peptide and a peptide of NUP153 bind the interface between the C-terminal and N- terminal domains of CA, indicating that this interface is a therapeutic target and may be important for docking of the capsid at the nuclear pore. We will now explore a surrogate of capsid docking at the nuclear pore by examination of 2D CA crystals with bound NUP153, allowing us to test whether the FG repeats bind cooperatively and whether binding disrupts the lattice to release the preintegration complex into the nucleus. We have shown that the R18F mutant of CA forms ~35 nm spherical particles, and a preliminary 3D cryoEM reconstruction shows that the ~90 Å spacing between protomers in the icosahedral lattice recapitulates the spacing in authentic HIV-1 capsids. We seek to determine an atomic resolution structure to examine the chemical interactions at the protomer-protomer interfaces. The 7-Å cryoEM map of mature HIV-1 and the high- resolution map of the icosahedral surrogate will enable computational simulations of the mature capsid lattice to investigate the effects of protein and drug binding on lattice self-assembly and stability. We believe our studies will yield novel insight into HIV-1 maturation and assembly that will be relevant for drug discovery.

抽象的我们研究计划的一个主题是准对称替代物的设计和组装 HIV-1 未成熟和成熟衣壳的六边形和多形晶格，可实现高分辨率通过 CryoEM、microED、X 射线晶体学和 ssNMR 光谱进行结构分析。通过添加多尺度计算模拟来强化实验结果。 · 目标 1：未成熟 Gag 晶格的结构研究：我们之前确定了 3.2 Å X 射线 CTD-SP1 Gag 构建体的结构，显示由 2 圈 CTD 和 2 圈组成的 6 螺旋束 SP1 的蛋白酶切割位点被隔离在束的内部。揭示了一个谜团：蛋白酶如何获得隔离的裂解位点？选择性标记的 ΔMA-Gag VLP 将使我们能够检查连接螺旋的构象动力学。 CTD-SP1 2D 晶体的时间分辨冷冻电镜（衍射分辨率为 5 Å）将测试蛋白酶是否裂解在 2-4 µm 3D 晶体的未成熟 Gag 晶格内的裂缝处开始。结合有 bevirimat 的 CTD-SP1 的 2.9 Å 分辨率 3D 结构，为我们探索奠定了基础我们的结果不仅会揭示第二代成熟抑制剂的耐药机制和作用。为原子分辨率 MD 模拟提供数据，还有助于开发和完善新型粗粒度 (CG) Gag 晶格组件模型，用于探索病毒颗粒的通路内和通路外生成的动力学。 · 目标 2：成熟衣壳的结构研究：我们之前使用二硫键交联来稳定并求解 CA 六聚体和五聚体的 X 射线结构所得富勒烯锥的原子模型。我们还表明了圆锥形衣壳曲率连续变化的机制。化合物 PF74、CPSF6 肽和 NUP153 的肽结合 C 端和 N 端之间的界面 CA 的末端结构域，表明该界面是治疗靶点，对于对接可能很重要我们现在将探索核孔处衣壳对接的替代物。检查结合了 NUP153 的 2D CA 晶体，使我们能够测试 FG 重复是否结合合作性以及结合是否破坏晶格以将预整合复合物释放到细胞核中。我们已经证明 CA 的 R18F 突变体形成约 35 nm 的球形颗粒，并且初步的 3D 冷冻电镜重建表明二十面体晶格中原聚体之间约 90 Å 的间距概括了我们寻求确定原子分辨率结构来检查真实的 HIV-1 衣壳中的间距。成熟 HIV-1 和高亲和力的 7-Å 冷冻电镜图。二十面体替代品的分辨率图将使成熟衣壳晶格的计算模拟成为可能研究蛋白质和药物结合对晶格自组装和稳定性的影响。研究将对 HIV-1 的成熟和组装产生新的见解，这将与药物发现相关。