Displaying functional molecules on plant virus-based nanoparticles

Plant virus nanoparticles (VNPs) have many unique advantages, including their ability to self-assemble with precise symmetry and polyvalency, their monodispersity and stability under a range of conditions, their inherent safety (biocompatibility and non-infectivity in mammals), and the rapid, simple and scalable production that can be achieved by molecular farming in plants. The structures of many plant viruses have been solved at atomic or near-atomic resolution and the genome sequences are well characterized, so plant VNPs can easily be modified to introduce new functions. However, selection for smaller genomes often causes the loss of transgenes during infection. This can be addressed by using the ribosomal skip (overcoat) strategy or chemical conjugation, although this reduces the density of peptides on the particle surface. This thesis describes new plant virus display systems for applications such as bone tissue engineering, bioimaging and plant biomass degradation. The first chapter describes Potato virus X (PVX) engineered to present mineralization-inducing peptides (MIPs) equivalent to non-collagenous proteins of the extracellular matrix. The mineralization of recombinant PVX particles was achieved using saturated hydroxyapatite solution and simulated body fluid, which closely mimic the mineral phase of human bone. These studies lay the foundations for the further exploitation of biomimetic VNPs in bone regeneration. The second chapter describes iLOV-displaying PVX and Tobacco mosaic virus (TMV) nanoparticles for bioimaging, which currently requires overcoating or chemical conjugation. Surprisingly, PVX particles with direct iLOV-coat protein (CP) fusions were infectious but this was not the case for TMV. The PVX particles may be useful for molecular imaging under low oxygen conditions, such as tumor metastasis, or for the analysis of particle behavior in hydrogels when combined with MIPs. A new strategy for protein display on TMV is also described, using a ribosomal skip sequence. Although not directly comparable, an eight-fold increase in the abundance of the displayed iLOV target protein was achieved compared to a malaria epitope fusion proteins created by using a read-through motif (Turpen et al., 1995), which may be advantageous in particular for vaccine production. The third chapter explores PVX and TMV as biocatalysts, using the protein-based attachment of endoglucanases as a case study. SpyTag/SpyCatcher technology allowed the rapid formation of a stable isopeptide bond between the enzyme and scaffold. The catalytic efficiency of flexible recombinant PVX particles was higher than that of rigid TMV particles, indicating that the morphology of the template influences catalytic activity. The results presented in this thesis provide new insights into PVX and TMV display systems and will be beneficial for many application areas by overcoming the limitations of genetic fusions, such as size constraints and the need for post-translational modifications