Collaborative Research: Engineering MS2 Capsid Assembly Using Systematic Fitness Landscapes

生物世界的大部分都是由被称为蛋白质的积木组成的，这些积木可以组装成肉眼看不到的复杂结构。这项提议的工作探索了一种由称为病毒样粒子的蛋白质组成的结构的自组装。了解病毒的组成部分是如何聚集在一起的，对于开发对抗病毒的治疗方法和疫苗可能很重要。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。推广工作将继续进行，包括本科生和研究生的参与和培训，通过在学校和科学节的演示，在当地图书馆和博物馆的讨论，与K-12学生和公众的参与，以及为6-12岁儿童制作和发布关于这项工作的顶级播客系列节目。病毒样颗粒是研究蛋白质自组装的一个很好的模型系统。这些闭壳蛋白容器往往由单一蛋白组成，可以在细菌中以高滴度表达。它们还具有封装自身遗传信息的有用特性。本研究利用这一特性，利用一种高效的定向进化新技术，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，该技术只需要一步就可以生成粒子文库，其中所有序列位置都被系统地替换为每个天然氨基酸。然后，这些文库可以暴露在各种选择压力下，通过选择幸存者中的封装RNA，能够识别具有所需物理或生化特性的颗粒。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，它将阐明噬菌体MS2衍生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，以便在任何期望的应用中进行修改后保持完整。该奖项反映了美国国家科学基金会的法定使命，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.