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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

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：

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

DOI：
10.1021/jacs.2c02568
发表时间：
2022-06-15
期刊：
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
影响因子：
15
作者：
Pak, Alexander J.;Gupta, Manish;Yeager, Mark;Voth, Gregory A.
通讯作者：
Voth, Gregory A.

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