Multimode Observation of Virus Capsid Assembly

摘要 乙肝病毒衣壳是由120个同源二聚体T=4组成的二十面体。在体内，它会自我组装， 包装病毒RNA，作为DNA合成的代谢室，在细胞内进行交易。 它的组装和拆解已成为有效抗病毒药物的靶标。在上一次筹资期间， 我们用结构、单分子和分子表征了纯化蛋白的组装和拆解。 对组装产品和反应的大量研究。我们对激活的变构转变进行了表征 组件，演示了在自关联期间可逆性对于保真度的重要性，并确定了 成核对引导组装路径的作用。 在这项提议中，我们提出了利用这些结果来设计病毒样颗粒的假设 具有可编程组装、货物包装、交付、投放等功能。在初步研究中，我们 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，基本上使用抗病毒作为靶向装置。这种方法可以应用于 任何货物。在某些情况下，希望在舱盖外部显示货物，在其他情况下，这是理想的 把它装进衣壳里。在初步数据中，我们演示了一种制造“孔状”衣壳的方法。 暴露在颗粒内部，并可以重新密封以封闭内容物。使用同样的技术， 我们可以在衣壳表面做补丁；这可以用来显示受体结合的补丁 配体或细胞穿透性多肽。包装在衣壳内的货物，除非它能 释放了。在初步数据中，我们开发了触发衣壳分解的技术，以响应 氧化还原电位，利用化学诱导的亚稳性。同样的方法也可以应用 至其他触发信号。这些研究的最终目标是将各种方法结合起来，以实现 结束：我们建议建立两个生物技术模型试剂，一个用于测量抗体水平，另一个用于 将包装好的货物送到特定的细胞。这些方法都建立在对 乙肝病毒衣壳组装的生物化学和生物物理。 乙肝病毒是人类最小的病原体之一。它在包装其基因组并将其传递给 将其发送到目标细胞。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 专门包装货物分子并在细胞内输送这些试剂的方法。这个 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。

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.