Structure Analysis of Viral Assembly Mechanisms

病毒组装机制的结构分析

• 批准号: 9926285
• 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
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9926285
• 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; Treatment Efficacy; 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; three dimensional structure

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.

摘要 我们的研究计划的一个主题是设计和组装对称的准- HIV-1未成熟和成熟衣壳的六角形和多晶型晶格，使高分辨率 通过低温电子显微镜、微电子能谱、X-射线结晶学和单核磁共振谱对其结构进行了分析。解读我们的 通过增加多尺度的计算模拟，实验结果得到了加强。 ·目标1：对未成熟的Gag晶格的结构研究：我们先前确定了一条3.2？X射线 CTD-SP1 Gag构建体的结构，它揭示了由2个CTD和2个旋转的CTD和2个螺旋组成的6-螺旋束 SP1的转数。蛋白水解酶的裂解部位被隔离在维管束内部。这一令人惊讶的结果 揭示了一个谜团：蛋白酶是如何进入被隔离的裂解位点的？单质核磁共振波谱 选择性标记的ΔMA-Gag VLP将使我们能够检测连接螺旋的构象动力学。 CTD-SP1的2D晶体的时间分辨低温电子显微镜，其衍射率为5？分辨率，将测试是否有蛋白酶 解理是在未成熟的Gag晶格内的裂隙处开始的。2-4微米3D晶体的微电火花加工产生了 结合Bevirimat的CTD-SP1的2.9维分辨率3D结构，为我们探索奠定了基础 耐药机制及第二代成熟抑制药的作用。我们的结果不仅会 为原子分辨率MD模拟提供数据，但也有助于开发和改进新的粗粒度(CG) GAG晶格组件的模型，以探索病毒粒子的路径上和路径外生成的动力学。 ·目标2：成熟衣壳的结构研究：我们以前曾使用二硫键交联来稳定 并解决了CA六聚体和五聚体的X射线结构。由此得到的富勒烯锥体的原子模型 提出了圆锥壳体中曲率连续变化的机制。我们还展示了 化合物PF74、CPSF6多肽和NUP153多肽结合C-端和N-端之间的界面。 CA的终端域，表明该界面是一个治疗靶点，可能对对接很重要 核孔处的衣壳。我们现在将探索衣壳对接在核孔处的替代方法 检查2D CA晶体与NUP153的结合，使我们能够测试FG是否重复结合 以及结合是否破坏晶格以将预整合复合体释放到核中。 我们已经证明了CA的R18F突变体形成了~35 nm的球形颗粒，并初步形成了三维低温电子显微镜 重建表明，二十面体晶格中原形分子之间的~90？间距重写了 正宗HIV-1衣壳中的间距。我们试图确定一个原子分辨率结构来检查 原-原界面上的化学相互作用。成熟的HIV-1和高度... 二十面体代理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。我们相信我们的 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。