Quantitative characterization of all single amino acid variants of a viral capsid-based drug delivery vehicle

Abstract

Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes—a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.

Unveiling the Hidden Rules of Spherical Viruses Using Point Arrays

“…These constraints can indicate where modifications are likely to succeed, although not what form they should take. We will see that in MS2, the gauge points reveal critical structural locations which are nearly impossible to modify [23,24].…”

Multiplexed Single Ion Mass Spectrometry Improves Measurement of Proteoforms and Their Complexes

“…When the OD600 reached 0.5, protein expression was induced with 0.1% arabinose. Expression continued overnight for ~ 16 h. After harvesting the cells by centrifugation, the cellular protein was extracted and VLPs were purified according to standard procedures using ammonium sulfate precipitation and fast protein liquid chromatography (FPLC) 24 .…”

“…Recently, these particles have been investigated through electron microscopy and X-ray crystallography to determine capsid structure [20][21][22][23][24] . Briefly, WT and MINI VLPs are composed of exactly 180 and 60 copies of the 13.7 kDa coat protein (CP), generating theoretical masses of 2.47 and 0.82 MDa, and particles with 27 and 17 nm diameters.…”

Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly

“…By contrast, human viruses often have reverse genetics systems in place where comprehensive mutant libraries can be prepared for single genes or regions within a gene (Doud et al 2018, Wu andQi 2019) using replicative plasmids that encode whole viruses or viral components. Similarly, deep mutational scanning can be performed on plasmid-encoded phage proteins like the MS2 capsid protein (Hartman et al 2018). As described by Hartman et al 2018, such experiments can identify mutations that confer useful characteristics, such as MS2 capsid stability mutations that could improve the targeted delivery of therapeutics to particular cellular compartments.…”

“…Similarly, deep mutational scanning can be performed on plasmid-encoded phage proteins like the MS2 capsid protein (Hartman et al 2018). As described by Hartman et al 2018, such experiments can identify mutations that confer useful characteristics, such as MS2 capsid stability mutations that could improve the targeted delivery of therapeutics to particular cellular compartments.…”