Plug-and-Display Photo-Switchable Systems on Plant Virus Nanoparticles.

Light can be used to regulate protein interactions with a high degree of spatial and temporal precision. Photo-switchable systems therefore allow the development of controllable protein complexes that can influence various cellular and molecular processes. Here, we describe a plant virus-based nanoparticle shuttle for the distribution of proteins that can be released when exposed to light. Potato virus X (PVX) is often used as a presentation system for heterologous proteins and epitopes, and has ideal properties for biomedical applications such as good tissue penetration and the ability to form hydrogels that present signaling molecules and promote cell adhesion. In this study, we describe three different systems attached to the surface of PVX particles: LOVTRAP, BphP1/QPAS1 and Dronpa145N. We demonstrated the functionality of all three photo-switchable protein complexes in vitro and the successful loading and unloading of PVX particles. The new systems provide the basis for promising applications in the biomedical and biomaterial sciences.

A targeted metabolomics method for extra- and intracellular metabolite quantification covering the complete monolignol and lignan synthesis pathway.

Microbial synthesis of monolignols and lignans from simple substrates is a promising alternative to plant extraction. Bottlenecks and byproduct formation during heterologous production require targeted metabolomics tools for pathway optimization. In contrast to available fractional methods, we established a comprehensive targeted metabolomics method. It enables the quantification of 17 extra- and intracellular metabolites of the monolignol and lignan pathway, ranging from amino acids to pluviatolide. Several cell disruption methods were compared. Hot water extraction was best suited regarding monolignol and lignan stability as well as extraction efficacy. The method was applied to compare enzymes for alleviating bottlenecks during heterologous monolignol and lignan production in E. coli. Variants of tyrosine ammonia-lyase had a considerable influence on titers of subsequent metabolites. The choice of multicopper oxidase greatly affected the accumulation of lignans. Metabolite titers were monitored during batch fermentation of either monolignol or lignan-producing recombinant E. coli strains, demonstrating the dynamic accumulation of metabolites. The new method enables efficient time-resolved targeted metabolomics of monolignol- and lignan-producing E. coli. It facilitates bottleneck identification and byproduct quantification, making it a valuable tool for further pathway engineering studies. This method will benefit the bioprocess development of biotransformation or fermentation approaches for microbial lignan production.

Advanced Fusion Strategies for the Production of Functionalized Potato Virus X Virions.

Plant virions are ideal for nanotechnology applications because they are structurally diverse and can self-assemble naturally, allowing for large-scale production in plants by molecular farming. Potato virus X (PVX) is particularly amenable due to the unique properties of its filamentous and flexible capsid, but efficient strategies are required to adapt the surface properties of PVX, such as the attachment of proteins and peptides. This chapter describes the selection and utilization of 2A ribosomal skip sequences, allowing the presentation of heterologous proteins and peptides as N-terminal fusions to the PVX coat protein at different densities. Another strategy for the rapid modification of PVX capsids is the plug-and-display module of the SpyTag/SpyCatcher system. The SpyTag can be presented on the PVX surface, allowing for the attachment of any protein fused to the SpyCatcher sequence.

From infection to healing: The use of plant viruses in bioactive hydrogels.

Plant viruses show great diversity in shape and size, but each species forms unique nucleoprotein particles that are symmetrical and monodisperse. The genetically programed structure of plant viruses allows them to be modified by genetic engineering, bioconjugation, or encapsulation to form virus nanoparticles (VNPs) that are suitable for a broad range of applications. Plant VNPs can be used to present foreign proteins or epitopes, to construct inorganic hybrid materials, or to carry molecular cargos, allowing their utilization as imaging reagents, immunomodulators, therapeutics, nanoreactors, and biosensors. The medical applications of plant viruses benefit from their inability to infect and replicate in human cells. The structural properties of plant viruses also make them useful as components of hydrogels for tissue engineering. Hydrogels are three-dimensional networks composed of hydrophilic polymers that can absorb large amounts of water. They are used as supports for tissue regeneration, as reservoirs for controlled drug release, and are found in contact lenses, many wound healing materials, and hygiene products. They are also useful in ecological applications such as wastewater treatment. Hydrogel-based matrices are structurally similar to the native extracellular matrix (ECM) and provide a scaffold for the attachment of cells. To fully replicate the functions of the ECM it is necessary to augment hydrogels with biological cues that regulate cellular interactions. This can be achieved by incorporating functionalized VNPs displaying ligands that influence the mechanical characteristics of hydrogels and their biological properties, promoting the survival, proliferation, migration, and differentiation of embedded cells. This article is categorized under: Implantable Materials and Surgical Technologies > Nanomaterials and Implants Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement.

Attachment of Ultralow Amount of Engineered Plant Viral Nanoparticles to Mesenchymal Stem Cells Enhances Osteogenesis and Mineralization.

Hydrogel-based materials are widely used to mimic the extracellular matrix in bone tissue engineering, although they often lack biofunctional cues. In the authors' previous work, Potato virus X (PVX), a flexible rod-shaped biocompatible plant virus nanoparticle (VNP) with 1270 coat protein subunits, is genetically modified to present functional peptides for generating a bone substitute. Here, PVX is engineered to present mineralization- and osteogenesis-associated peptides and laden in hydrogels at a concentration lower by two orders of magnitude. Its competence in mineralization is demonstrated both on 2D surfaces and in hydrogels and the superiority of enriched peptides on VNPs is verified and compared with free peptides and VNPs presenting fewer functional peptides. Alkaline phosphatase activity and Alizarin red staining of human mesenchymal stem cells increase 1.2-1.7 times when stimulate by VNPs. Engineered PVX adheres to cells, exhibiting a stimulation of biomimetic peptides in close proximity to the cells. The retention of VNPs in hydrogels is monitored and more than 80% of VNPs remain inside after several washing steps. The mechanical properties of VNP-laden hydrogels are investigated, including viscosity, gelling temperature, and compressive tangent modulus. This study demonstrates that recombinant PVX nanoparticles are excellent candidates for hydrogel nanocomposites in bone tissue engineering.

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

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.

In Planta Production of Fluorescent Filamentous Plant Virus-Based Nanoparticles.

Viral nanoparticles are attractive platforms for biomedical applications and are frequently employed for optical imaging in tissue culture and preclinical animal models as fluorescent probes. Chemical modification with organic dyes remains the most common strategy to develop such fluorescent probes. Here we report a genetic engineering approach to incorporate fluorescent proteins in viral nanoparticles, which can be propagated in their plant host. The fluorescent viral nanoparticles so obtained obviate post-harvest modifications and thereby maximize yields. Our engineering approach transforms filamentous potato virus X (PVX) to display green fluorescent protein (GFP) or mCherry as N-terminal coat protein (CP) fusions at a 1:3 fusion protein to CP ratio through integration of the foot-and-mouth disease 2A sequence. The in planta propagation of recombinant GFP-PVX or mCherry-PVX thus produced in Nicotiana benthamiana can be easily documented using fluorescence imaging. Molecular farming protocols can be accordingly optimized by monitoring chimera stability over the course of the infection cycle. Moreover, we also demonstrate the utility of recombinant mCherry-PVX in optical imaging of human cancer cells and tumor tissue in preclinical mice model. Together, these features make genetically engineered fluorescent PVX particles ideally suited for molecular imaging applications.

Bioinspired Silica Mineralization on Viral Templates.

Plant virus capsids are attractive entities for nanotechnological applications because of their variation in shape and natural assembly ability. This chapter describes the production and modification of three differently shaped plant virus capsids for silica mineralization purposes. The chosen plant viruses exhibit either an icosahedral (cowpea mosaic virus, CPMV), or a flexuous rod-like structure (potato virus X, PVX), or a rigid rod-like shape (tobacco mosaic virus, TMV), and are well-known and frequently used plant viruses for biotechnological applications. We describe the production (including genetic or chemical modification) and purification of the plant viruses or of empty virus-like particles in the case of CPMV, as well as the characterization of these harvested templates. The mineralization procedures and differences in the protocols specific to the distinct viruses are described, and the analyses of the mineralization results are explained.

Systemic Infection of Nicotiana benthamiana with Potato virus X Nanoparticles Presenting a Fluorescent iLOV Polypeptide Fused Directly to the Coat Protein.

Plant virus-based nanoparticles can be produced in plants on a large scale and are easily modified to introduce new functions, making them suitable for applications such as vaccination and drug delivery, tissue engineering, and in vivo imaging. The latter is often achieved using green fluorescent protein and its derivatives, but the monovalent fluorescent protein iLOV is smaller and more robust. Here, we fused the iLOV polypeptide to the N-terminus of the Potato virus X (PVX) coat protein, directly or via the Foot-and-mouth disease virus 2A sequence, for expression in Nicotiana benthamiana. Direct fusion of the iLOV polypeptide did not prevent the assembly or systemic spread of the virus and we verified the presence of fusion proteins and iLOV hybrid virus particles in leaf extracts. Compared to wild-type PVX virions, the PVX particles displaying the iLOV peptide showed an atypical, intertwined morphology. Our results confirm that a direct fusion of the iLOV fluorescent protein to filamentous PVX nanoparticles offers a promising tool for imaging applications.

Engineered Potato virus X nanoparticles support hydroxyapatite nucleation for improved bone tissue replacement.

Bionanoparticles based on filamentous phages or flexuous viruses are interesting candidates for meeting the challenges of tailoring biomineralization in hydrogel-based bone tissue substitutes. We hypothesized that hydroxyapatite crystal nucleation and matrix mineralization can be significantly increased by mineralization-inducing (MIP) and integrin binding motif (RGD) peptides presented on biomimetic nanoparticles. In this study, Potato virus X (PVX), a flexible rod-shaped plant virus was genetically engineered to present these functional peptides on its particle surface. Recombinant PVX-MIP/RGD particles were isolated from infected Nicotiana benthamiana plants and characterized by western blot, SEM, TEM, and TPLSM in MSC cultures. The presence of RGD was proven by cell attachment, spreading, and vinculin cluster analysis, and MIP by in vitro mineralization and osteogenic differentiation assays. Thus the tailored surface of genetically engineered PVX forms fibril-like nanostructures which enables enhanced focal adhesion-dependent cell adhesion, and matrix mineralization verified by Alizarin. Hydroxyapatite crystal nucleation is supported on recombinant PVX particles leading to a biomimetic network and bundle-like structures similar to mineralized collagen fibrils. In conclusion, the recombinant flexuous PVX nanoparticles exhibit properties with great potential for bone tissue substitutes. STATEMENT OF SIGNIFICANCE: A suitable biomaterial for tissue engineering should be able to mimic the endogenous extracellular matrix by presenting biochemical and biophysical cues. Novel hydrogel-based materials seek to meet the criteria of cytocompatibility, biodegradability, printability, and crosslinkability under mild conditions. However, a majority of existing hydrogels lack cell-interactive motifs, which are crucial to modulate cellular responses. The incorporation of the plant virus PVX to the hydrogel could improve functions like integrin-binding and mineralization due to peptide-presentation on the particle surface. The tailored surface of genetically engineered PVX forms fibril-like nanostructures which enables enhanced focal adhesion-dependent cell adhesion and matrix mineralization and offers great potential for the development of new hydrogel compositions for bone tissue substitutes.

Production of Hybrid Chimeric PVX Particles Using a Combination of TMV and PVX-Based Expression Vectors.

We have generated hybrid chimeric potato virus X (PVX) particles by coexpression of different PVX coat protein fusions utilizing tobacco mosaic virus (TMV) and PVX-based expression vectors. Coinfection was achieved with a modified PVX overcoat vector displaying a fluorescent protein and a TMV vector expressing another PVX fluorescent overcoat fusion protein. Coexpression of the PVX-CP fusions in the same cells was confirmed by epifluorescence microscopy. Labeling with specific antibodies and transmission electron microscopy revealed chimeric particles displaying green fluorescent protein and mCherry on the surface. These data were corroborated by bimolecular fluorescence complementation. We used split-mCherry fragments as PVX coat fusions and confirmed an interaction between the split-mCherry fragments in coinfected cells. The presence of assembled split-mCherry on the surface confirmed the hybrid character of the chimeric particles.

Potato virus X-based expression vectors are stabilized for long-term production of proteins and larger inserts.

Plus-strand RNA viruses such as Potato virus X (PVX) are often used as high-yielding expression vectors in plants, because they tolerate extra transgene insertion and expression without disrupting normal virus functions. However, sequence redundancy due to promoter duplication often leads to genetic instability. Although heterologous subgenomic promoter-like sequences (SGPs) have been successfully used in Tobacco mosaic virus vectors, only homologous SGP duplications have been used in PVX vectors. We stabilized PVX-based vectors by combining heterologous SGPs from related potexviruses with an N-terminal coat protein (CP) deletion. We selected two SGPs with core sequences homologous to PVX, from Bamboo mosaic virus (BaMV) and Cassava common mosaic virus, as well as a SGP with a heterologous core sequence from Foxtail mosaic virus (FoMV). We found that only the BaMV and CsCMV SGPs were utilized by the PVX replicase. However, the transgene remained unstable, due to the presence of an additional region with strong sequence similarity at the 5' end of the cp gene. The BaMV SGP combined with an N-terminal CP deletion achieved high PVX vector stability. This new expression vector is particularly useful for long-term production of proteins and for larger inserts. The improved PVX-based vectors are suitable for the systemic expression of any gene of interest in PVX host plants. The PVX-based vector can be advantageous for the overexpression of proteins, to analyze protein functions in planta or as a system for virus-induced gene silencing.