Collaborative Research: Engineering MS2 Capsid Assembly Using Systematic Fitness Landscapes

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.