title: Templated Assembly of Virus-Like Particles
Creators: name: McNeale, Donna
Publisher: name: Griffith University
Year: 2023
Subjects: subject: virus
subject: capsid
subject: self-assembly

description: Owing to their remarkable abundance and diversity, viruses, and by extension virus-like particles (VLPs), have gained increasing attention for their applications in bionanotechnology. The structural proteins of VLPs self-assemble into highly symmetrical, uniform architectures. The ability to control the self-assembly and selforganisation of molecules at the nanoscale is a fundamental principle underlying the broad range of applications of bionanotechnology, from materials to medicine. In the work presented here, the use of templating to mediate assembly and impart new functionalities on two different VLP systems is explored. Bacteriophage P22-based VLPs are exploited for their use as a size-constrained vessel for the confinement of reactions. P22 VLPs are built from two components: coat protein (CP) and scaffold protein (SP). Co-expression of CP and SP in bacteria leads to in vivo assembly, and encapsulation of protein cargos is achieved via fusion to SP. The SP-cargo fusion functions as a template for the assembly of CP thereby mediating encapsulation. By utilising a dual plasmid system for the individual expression of multiple SP-cargos, in vivo co-encapsulation was achieved. Moreover, temporal control of the induction of the expression of two SP-cargo fusions enabled control over cargo stoichiometry and density. The successful coencapsulation of two different fluorescent proteins was confirmed using Förster resonance energy transfer. A two-step enzymatic reaction was also successfully coencapsulated within P22-VLPs with various enzyme stoichiometries and loading densities. This produced a range of differentially loaded nanoreactors for the synthesis of a pharmaceutically relevant unnatural amino acid and analysis of reaction products revealed an impact of enzyme stoichiometry and loading density on enzyme activity, demonstrating the utility of this method in the creation and optimisation of efficient P22- based nanoreactors. In contrast to the robust structure of P22, the flexibility of murine polyomavirus (MPyV) VLPs can be exploited for the assembly of morphologically distinct hybrid nanoparticles. MPyV VLPs can be assembled from a single protein, VP1, which is isolated in its pentameric form following bacterial expression. VP1 assembly into empty VLPs can be triggered in vitro. The ability to bind dsDNA was confirmed and the assembly of VP1 in the presence of DNA suggested that this did not interfere with in vitro assembly of VLPs. Moreover, the presence of DNA did not trigger VLP assembly. However, given the correct conditions, the interactions between DNA and VP1 directed assembly towards morphologies that mirrored the DNA template. The capacity to form cargo-dependent morphologies was used for the creation of three-dimensional DNAprotein hybrid structures. Under the same conditions, VP1 was templated onto three different sizes of icosahedral DNA origami wireframes. The size of the resultant particles implied that the assembly of VP1 was constrained by the DNA origami template. Furthermore, the templated VLPs exhibited tighter size distributions compared to empty VLPs. Cryo-electron microscopy analysis of one of the templated VLPs revealed that the icosahedral symmetry of the capsid was retained. The DNA origami was regularly positioned within the VLP, with the symmetries of both the protein shell and DNA origami aligned. Densities potentially representing the N-termini of VP1 connected the protein shell with the vertices and edges of the DNA origami, suggesting assembly is driven by interactions between VP1 and the DNA origami. Theoretical models on templated assembly indicate that assembly requires a balance between the relative strength of protein-protein and protein-DNA interactions, and for the case of the DNA origami templated VLPs, it appeared that the strength of these interactions was permissive to efficient VLP assembly. The work presented in this thesis demonstrates the utility of templated assembly of VLPs with protein scaffolds and DNA wireframes to construct functional hybrid nanostructures. These hybrid nanostructures serve as tools to help expand our understanding of virus capsid assembly and highlights their potential in biocatalysis and gene delivery.