Collaborative Research: Engineering MS2 Capsid Assembly Using Systematic Fitness Landscapes

生物世界的大部分是由称为蛋白质的构建模块组成的，它们可以以肉眼不可见的尺度组装成复杂的结构。这项拟议中的工作探索了一种由蛋白质组成的结构的自组装，这种结构被称为病毒样颗粒。了解病毒的组成部分如何聚集在一起，对于开发治疗方法和疫苗来对抗它们非常重要。这项工作的目的是揭示这种病毒颗粒如何组装的规则，以及如何在不破坏其结构的情况下对其进行修改以改善其功能。将继续开展外联工作，包括本科生和研究生的参与和培训，以及通过在学校和科学节上的演示，在当地图书馆和博物馆的讨论，并为6- 12岁的儿童创作和发布一集关于这项工作的顶级播客系列。病毒-类粒子是研究蛋白质自组装的理想模型系统。这些闭壳蛋白容器倾向于由单一蛋白质组成，并且可以在细菌中表达至高滴度。它们还具有封装自身遗传信息的有用特性。该建议利用这一特性，使用一种有效的新的定向进化技术来研究突变对病毒样颗粒自组装特性的有利和有害影响。重要的是，该技术使用一个步骤来生成颗粒库，其中所有序列位置都被每个天然氨基酸系统地取代。然后可以将这些文库暴露于各种选择压力，使得能够通过选择的幸存者中的包封RNA鉴定具有期望的物理或生物化学性质的颗粒。利用各种诱变和功能选择，这项工作旨在实现两个具体目标。首先，它将阐明来自噬菌体MS 2的病毒样颗粒组装的关键要求。其次，它将提高颗粒的化学反应性和稳定性，因此在任何所需的应用中，它都可以在修改后保持完整。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Much of the biological world is made up building blocks, known as proteins, which can assemble into complex structures at scales not visible to the naked eye. The proposed work explores the self-assembly of a structure made of proteins called a virus-like particle. Understanding how the building blocks of viruses come together could be important for developing therapeutics and vaccines to fight them. The objective of this work is to uncover the rules for how this virus particle assembles and how it can be modified to improve its function without disrupting its structure. Outreach efforts will be pursued that include engagement and training of undergraduate and graduate students, and engagement with K-12 students and the general public via demonstrations in schools and at science festivals, discussions at local libraries and museums, and creation and release of an episode about this work on a top-ranked podcast series for children ages 6-12.Virus-like particles are an excellent model system to study protein self-assembly. These closed-shell protein containers tend to be composed of a single protein and can be expressed to high titers in bacteria. They also have the useful property of encapsulating their own genetic information. This proposal takes advantage of this property to study both the advantageous and deleterious effects of mutations on the self-assembly properties of virus-like particles using an efficient new directed evolution technique. Importantly, this technique uses a single step to generate libraries of the particles in which all sequence positions are systematically substituted with every natural amino acid. These libraries can then be exposed to various selection pressures, enabling identification of particles with desirable physical or biochemical properties via the encapsulated RNA in survivors of the selection. Using a variety of mutagenesis and functional selections, this work aims to achieve two specific objectives. First, it will elucidate the critical requirements for assembly of the virus-like particle derived from bacteriophage MS2. Second, it will improve the chemical reactivity and stability of the particle so it can remain intact following modifications for any desired application.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.