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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.

摘要我们的研究计划的一个主题是设计和组装对称的替代品的准， HIV-1未成熟和成熟衣壳的六边形和多晶晶格，通过cryoEM、microED、X射线晶体学和ssNMR光谱进行结构分析。我们的解释实验结果通过增加多尺度的计算模拟得到加强。 ·目标1：未成熟Gag晶格的结构研究：我们先前确定了3.2 μ m的X射线衍射。 CTD-SP1 Gag构建体的结构，其揭示了由2个CTD转角和2个螺旋组成的6螺旋束。 SP1的旋转。蛋白酶切割位点被隔离在束的内部。这个令人惊讶的结果揭示了一个谜团：蛋白酶如何进入隔离的切割位点？ssNMR光谱学选择性标记的ΔMA-Gag VLP将使我们能够检查连接螺旋的构象动力学。 CTD-SP1二维晶体的时间分辨cryoEM，其分辨率可达5 μ m，将测试蛋白酶解理在未成熟Gag晶格内的裂缝处开始。2-4 µm 3D晶体的MicroED产生了结合贝韦瑞马特的CTD-SP1的2.9 Å分辨率3D结构，为我们的探索奠定了基础耐药机制和第二代成熟抑制剂的作用。我们的成果不仅为原子分辨率的MD模拟提供数据，但也有助于开发和改进新的粗粒度（CG） Gag晶格组分的模型，以探索病毒颗粒的通路上和通路外生成的动力学。 ·目标2：成熟衣壳的结构研究：我们先前使用二硫键交联来稳定衣壳。并解出CA六聚体和五聚体的X射线结构。由此产生的富勒烯锥的原子模型建议机制的连续变化的曲率在锥形衣壳。我们还表明，化合物PF 74、CPSF 6肽和NUP 153肽结合C-末端和N-末端之间的界面。 CA的末端结构域，表明该界面是治疗靶点，并且对于对接可能是重要的在核孔处。我们现在将探索衣壳对接在核孔的替代物，检查具有结合NUP 153的2D CA晶体，允许我们测试FG重复是否结合合作以及结合是否破坏晶格以将整合前复合物释放到细胞核中。我们已经表明，CA的R18 F突变体形成约35 nm的球形颗粒，并且初步的3D cryoEM 重建表明，在二十面体晶格中，原聚体之间的~90 μ m间距概括了真正的HIV-1衣壳中的间隔。我们试图确定一个原子分辨率结构来检查原聚体-原聚体界面的化学相互作用。成熟的HIV-1和高表达的HIV-1的7-kDa cryoEM图谱，二十面体替代物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。我们相信我们的这些研究将对HIV-1的成熟和组装产生新的见解，这将与药物发现有关。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Preservation of HIV-1 Gag Helical Bundle Symmetry by Bevirimat Is Central to Maturation Inhibition.

DOI：
10.1021/jacs.1c08922
发表时间：
2021-11-17
期刊：
Journal of the American Chemical Society
影响因子：
15
作者：
Pak AJ;Purdy MD;Yeager M;Voth GA
通讯作者：
Voth GA

Critical mechanistic features of HIV-1 viral capsid assembly.

DOI：
10.1126/sciadv.add7434
发表时间：
2023-01-06
期刊：
Science advances
影响因子：
13.6
作者：
通讯作者：

Immature HIV-1 assembles from Gag dimers leaving partial hexamers at lattice edges as potential substrates for proteolytic maturation.

DOI：
10.1073/pnas.2020054118
发表时间：
2021-01-19
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Tan A;Pak AJ;Morado DR;Voth GA;Briggs JAG
通讯作者：
Briggs JAG

Inositol Hexakisphosphate (IP6) Accelerates Immature HIV-1 Gag Protein Assembly toward Kinetically Trapped Morphologies.