Engineering a Virus-like Particle to Display Peptide Insertions Using an Apparent Fitness Landscape

Peptide
insertions in the primary sequence of proteins expand functionality
by introducing new binding sequences, chemical handles, or membrane
disrupting motifs. With these properties, proteins can be engineered
as scaffolds for vaccines or targeted drug delivery vehicles. Virus-like
particles (VLPs) are promising platforms for these applications since
they are genetically simple, mimic viral structure for cell uptake,
and can deliver multiple copies of a therapeutic agent to a given
cell. Peptide insertions in the coat protein of VLPs can increase
VLP uptake in cells by increasing cell binding, but it is difficult
to predict how an insertion affects monomer folding and higher order
assembly. To this end, we have engineered the MS2 VLP using a high-throughput
technique, called Systematic Mutagenesis and Assembled Particle Selection
(SyMAPS). In this work, we applied SyMAPS to investigate a highly
mutable loop in the MS2 coat protein to display 9,261 non-native tripeptide
insertions. This library generates a discrete map of three amino acid
insertions permitted at this location, validates the FG loop as a
valuable position for peptide insertion, and illuminates how properties
such as charge, flexibility, and hydrogen bonding can interact to
preserve or disrupt capsid assembly. Taken together, the results highlight
the potential to engineer VLPs in a systematic manner, paving the
way to exploring the applications of peptide insertions in biomedically
relevant settings.