A Selection for Assembly Reveals That a Single Amino Acid Mutant of the Bacteriophage MS2 Coat Protein Forms a Smaller Virus-like Particle

Abstract

Virus-like particles are used to encapsulate drugs, imaging agents, enzymes, and other biologically active molecules in order to enhance their function. However, the size of most virus-like particles is inflexible, precluding the design of appropriately sized containers for different applications. Here, we describe a chromatographic selection for virus-like particle assembly. Using this selection, we identified a single amino acid substitution to the coat protein of bacteriophage MS2 that mediates a uniform switch in particle geometry from T = 3 to T = 1 icosahedral symmetry. The resulting smaller particle retains the ability to be disassembled and reassembled in vitro and to be chemically modified to load cargo into its interior cavity. The pair of 27 and 17 nm MS2 particles will allow direct examination of the effect of size on function in established applications of virus-like particles, including drug delivery and imaging.

Bacteriophage MS2 displays unreported capsid variability assembling T = 4 and mixed capsids

“…Hence, the genome of MS2 is thought to restrict the folding pathway to select for the assembly of physiological T = 3 capsids, preventing the formation of other capsid forms (Dai et al, 2017). Previous studies identified both smaller and larger non-infectious MS2 particles formed in vitro (Sugiyama et al, 1967), however, the architecture of these non-standard capsids (mostly likely mixed T = 1, T = 3 and T = 3, T = 4 structures given our results and those of Asensio et al, 2016) and why they might be problematic for infection remained unknown.…”

“…On the observation of divergent symmetry VLPs, we created an otherwise identical wild-type (wt) construct with monomeric CP and prepared cryo-EM grids to confirm or deny that the presence of the CP-His-CP dimer was not solely responsible for the formation of mixed and large capsids as previously reported for non-wild-type constructs (Peabody and Chakerian, 1999;Plevka et al, 2008Plevka et al, , 2009Asensio et al, 2016;Zhao et al, 2019). We confirmed similar observations in the micrographs of the wild-type setting; in the higher molecular weight fractions of the wt-CP sample, the majority of the particles belonged to the smaller class, ~85% of the total on sorting by 2D classification, while several varieties of 'large' particles accounted for the remaining ~15%, of which ~6% later proved to be large icosahedral particles ( Supplementary Fig.…”

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

“…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.…”

“…3d) species. The lack of distinct charge states in the native MS data was attributed to the molecular heterogeneity of varying lengths of DNA and mRNA 22 . Given this molecular heterogeneity and no distinct charge state peaks present in the m/z-domain spectra, no information regarding mass of the VLP plus cargo could be discerned.…”

“…Although the length of the DNA base pairs and mRNA nucleotides that form the VLP cargo loads vary across a wide distribution of lengths, MINI VLPs having a much smaller volume incorporate shorter DNA and mRNA strands than their WT counterparts. DNA and mRNA lengths averaged 900 (±400) vs. 375 (±150) base pairs (bp) and 500 (±250) vs. 100 (±50) nucleotides (nt) for WT vs. MINI species 22 . The mass distribution for the WT particles determined via hCDMS ( Fig.…”