Adapting the Interior and Exterior Cargo of Barley-Stripe Mosaic Virus-Like Particles

• Jesal Patel, Biochemistry, University of Delaware

• Kevin Solomon, Chemical & Biomolecular Engineering, University of Delaware

Nanoparticles are becoming increasingly significant in areas such as sensing, catalysis, and medicine. Rod-shaped plant viruses, like barley stripe mosaic virus (BSMV), exhibit beneficial properties such as biocompatibility, scalable production, and a monodisperse hierarchical structure. Moreover, the viral particle surfaces offer numerous sites for the attachment of functional ligands, including small chemicals, metals, and biopolymers. By replacing their viral genomes with custom RNA templates, these viruses can form non-infectious virus-like particles (VLPs) with modular cargo. However, achieving precise control over VLP length, aspect ratio, and composition is critical for designing VLPs tailored for specific applications. We leverage a bacterial platform for BSMV VLP production to vary RNA cargo length and generate nanorods with controllable lengths. We successfully created VLPs of increasing sizes by varying the RNA template lengths, observing a proportional relationship between RNA template size and VLP size as confirmed by dynamic light scattering (DLS). We are currently working on testing additional RNA template lengths to further optimize VLP size and functionality. 

Controlling the surface cargo of BSMV VLPs is also critical for specific applications. Direct fusion to the surface of these VLPs are shown to be effective for peptides, but larger proteins require extensive optimization. To address this, we are incorporating SpyCatcher, a bacterial protein that binds specifically and irreversibly to the short peptide SpyTag. This approach will create a highly modular platform that can be easily functionalized with a variety of SpyTagged ligands. We successfully cloned BSMV to SpyCatcher at four insertion sites via Gibson assembly. Despite observing cleavage of the Spycatcher fusion, the VLPs still assembled. We are now screening other insertions sites to remove the cleavage site. These advancements enable precise control over BSMV VLP size and composition, enhancing their potential for various applications.