Multimode Observation of Virus Capsid Assembly

病毒衣壳组装的多模式观察

基本信息

批准号：
10587218
负责人：
Adam Zlotnick
金额：
$ 49.34万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-14 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10587218
关键词：
Address Affinity Allosteric Regulation Antibodies Antiviral Agents Behavior Binding Biochemistry Biological Assay Biophysics Biotechnology Capsid Capsid Proteins Cells Charge Chemicals Chemistry Chronic Chronic Hepatitis B Complex Crowding Cytoplasm DNA biosynthesis Data Detection Development Devices Dissociation Endosomes Engineering Environment Epitopes Exposure to Funding Genome Goals Growth Hepatitis B Virus Kinetics Learning Life Cycle Stages Ligand Binding Link Mass Spectrum Analysis Measures Metabolic Methods Modeling Modification Molecular Biology Oxidation-Reduction Paper Penetration Peptides Proteins RNA Reaction Reagent Research Roentgen Rays Role Self Correction Signal Transduction Structure Surface System Techniques Technology Testing Text Thermodynamics Time Variant Viral Viral Genome Viral Packaging Virus Virus Assembly Virus Diseases Virus-like particle delivery vehicle dimer falls flexibility human pathogen improved in vivo macromolecule man particle exposure practical application preservation protein purification receptor receptor binding response seal self assembly single molecule small molecule tool viral RNA

项目摘要

Summary The capsid of the Hepatitis B Virus (HBV) is a 120-homodimer T=4 icosahedron. In vivo, it self-assembles, packages viral RNA, serves as a metabolic compartment for DNA synthesis, and trafficks within the cell. Its assembly and disassembly have become targets for potent antivirals. In the previous funding period, we characterized assembly and disassembly with purified protein using structural, single molecule, and bulk studies of assembly products and reactions. We characterized the allosteric transitions that activated assembly, demonstrated the importance of reversibility during self-association for fidelity, and identified roles of nucleation for directing the assembly path. In this proposal, we develop hypotheses to take advantage of these results to engineer virus-like particles with programmable assembly, cargo packaging, delivery, and release. In preliminary studies, we developed a method for targeting cargo to a capsid by linking the cargo to a small molecule that binds capsid with high affinity, essentially using an antiviral as a targeting device. This method can be applied to any cargo. In some cases, it is desirable to display cargo on the capsid exterior, in other cases it is desirable to package it within the capsid. In preliminary data, we demonstrate an approach to making “holey” capsids that expose the particle interior and can be re-sealed to enclose the contents. Using this same technology, we can make patches on the capsid surface; this can be used for displaying patches of receptor-binding ligands or cell-penetrating peptides. Cargo, packaged within a capsid, is not deliverable unless it can be released. In preliminary data, we developed techniques for triggering capsid disassembly in response to redox potential, taking advantage of chemically-induced metastability. This same approach can be applied to other triggering signals. The ultimate goal of these studies is to combine the approaches to a practical end: we propose to build two model biotech reagents, one to measure antibody levels and the other to deliver packaged cargo to specific cells. These approaches are each built on an understanding of the biochemistry and biophysics of HBV capsid assembly. HBV is one the smallest human pathogens. It is remarkably efficient at packaging its genome and delivering it to target cells. Based on our understanding of capsid assembly and capsid biophysics, we will develop approaches to specifically packaging cargo molecules and delivering these reagents intracellularly. The tools arising from this research will provide a means for man to take advantage of HBV.

概括乙型肝炎病毒 (HBV) 的衣壳是 120 同型二聚体 T=4 二十面体，在体内自我组装，包装病毒RNA，作为DNA合成的代谢区室，并在细胞内运输。在之前的资助期间，它的组装和分解已成为强效抗病毒药物的目标。我们使用结构、单分子和纯化蛋白质来表征组装和拆卸我们对组装产物和反应的大量研究描述了激活的变构转变。组装，证明了自关联过程中可逆性对于保真度的重要性，并确定了成核在指导组装路径中的作用。在本提案中，我们提出假设，利用这些结果来设计病毒样颗粒在初步研究中，我们通过可编程组装、货物包装、交付和释放。开发了一种通过将货物连接到结合的小分子来将货物靶向衣壳的方法衣壳具有高亲和力，本质上可以使用抗病毒药物作为靶向装置。在某些情况下，需要在衣壳外部展示货物，而在其他情况下则需要。将其包装在衣壳中在初步数据中，我们演示了一种制作“多孔”衣壳的方法。暴露颗粒内部并可以使用相同的技术重新密封以封装内容物，我们可以在衣壳表面制作补丁；这可以用于显示受体结合的补丁包装在衣壳内的配体或细胞穿透肽除非可以被递送，否则是不可递送的。在初步数据中，我们开发了触发衣壳分解的技术。氧化还原电位，利用化学诱导的亚稳定性可以应用相同的方法。这些研究的最终目标是将这些方法结合到实际中。结束：我们建议构建两种模型生物技术试剂，一种用于测量抗体水平，另一种用于测量抗体水平将包装好的货物运送到特定的细胞。这些方法都是建立在对乙型肝炎病毒衣壳组装的生物化学和生物物理学。乙型肝炎病毒是最小的人类病原体之一，它在包装基因组和传递基因方面非常有效。基于我们对衣壳组装和衣壳生物物理学的理解，我们将开发它。特异性包装货物分子并在细胞内递送这些试剂的方法。这项研究产生的工具将为人类利用乙肝病毒提供一种手段。

项目成果

期刊论文数量（25）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids.

DOI：
10.7554/elife.31473
发表时间：
2018-01-29
期刊：
eLife
影响因子：
7.7
作者：
Schlicksup CJ;Wang JC;Francis S;Venkatakrishnan B;Turner WW;VanNieuwenhze M;Zlotnick A
通讯作者：
Zlotnick A

The Structural Biology of Hepatitis B Virus: Form and Function.