Small, Smaller, Nano: New Applications for Potato Virus X in Nanotechnology

Abstract

Nanotechnology is an expanding interdisciplinary field concerning the development and application of nanostructured materials derived from inorganic compounds or organic polymers and peptides. Among these latter materials, proteinaceous plant virus nanoparticles have emerged as a key platform for the introduction of tailored functionalities by genetic engineering and conjugation chemistry. Tobacco mosaic virus and Cowpea mosaic virus have already been developed for bioimaging, vaccination and electronics applications, but the flexible and filamentous Potato virus X (PVX) has received comparatively little attention. The filamentous structure of PVX particles allows them to carry large payloads, which are advantageous for applications such as biomedical imaging in which multi-functional scaffolds with a high aspect ratio are required. In this context, PVX achieves superior tumor homing and retention properties compared to spherical nanoparticles. Because PVX is a protein-based nanoparticle, its unique functional properties are combined with enhanced biocompatibility, making it much more suitable for biomedical applications than synthetic nanomaterials. Moreover, PVX nanoparticles have very low toxicity in vivo, and superior pharmacokinetic profiles. This review focuses on the production of PVX nanoparticles engineered using chemical and/or biological techniques, and describes current and future opportunities and challenges for the application of PVX nanoparticles in medicine, diagnostics, materials science, and biocatalysis.

Keywords: bioinspired materials; chemical conjugation; drug delivery; genetic engineering; imaging; nanoparticles; plant virus.

FIGURE 1

Potato virus X particles. Transmission electron micrograph and schematic representation of PVX. Scale bar = 500 nm. Adapted from Lauria et al. (2017).

FIGURE 2

Schematic representation of modifications evaluated for the production of PVX-based VNPs. Certain drugs (blue hexagons) such as doxorubicin can intercalate between the coat protein (CP) subunits of particles via hydrophobic interactions (Le et al., 2017b; reproduced by permission of The Royal Society of Chemistry). By genetic engineering, it is possible to fuse a target sequence to the 5′-end of the cp gene as a direct fusion, with an additional linker, or with an intervening 2A sequence. This strategy leads to the production of particles carrying the protein of interest (red) on every CP copy as direct fusions or on ∼25% of the CP copies with the 2A sequence. The fusion of a SpyTag peptide (dark red) to the particle surface makes it possible to attach any protein of interest (purple) fused to a SpyCatcher protein (light blue) expressed in any system. The PVX particle surface also features reactive groups (bright green) offered by lysine and cysteine residues that can be used for the chemical coupling of target molecules (R₁). N-hydroxysuccinimide or maleimide chemistry is typically used for this purpose. For the successful presentation of several large proteins utilizing the 2A sequence (yellow), a second set of a PVX CP fusion constructs (blue) can be co-expressed in the same cell by co-infection with a TMV vector. 2A, Foot-and-mouth disease virus 2A sequence. CaMV 35S/pA35S, Cauliflower mosaic virus 35S promoter/polyadenylation sequence. CP, coat protein. GOI, gene of interest. L, linker. MP, movement protein. Small arrows indicate subgenomic promoter-like sequences. Adapted from Le et al. (2017b).

FIGURE 3

Applications of PVX nanoparticles. PVX has been evaluated for diverse applications including molecular imaging, tumor homing and drug delivery, vaccination, biosensor design, biomaterials development, and biocatalysis. Adapted from Röder et al. (2017).

FIGURE 4

PVX for imaging applications. (A) GFP-labeled CP and mCherry-labeled TGBp1 for subcellular localization. Reproduced with permission from Tilsner et al. (2012). Scale bar = 10 μm. (B) Biodistribution of PVX-mCherry particles within the organs and tumors of mice (Shukla et al., 2014a). (C) Infection of N. benthamiana with PVX carrying iLOV and (D) localization of iLOV-CP direct fusion proteins within plasmodesmata (Röder et al., 2018). PD, plasmodesmata. VCR, viral replication complex. Adapted from Tilsner et al. (2012), Shukla et al. (2014a), and Röder et al. (2018).