A Combination of Flexible Modified Plant Virus Nanoparticles Enables Additive Effects Resulting in Improved Osteogenesis.

Plant virus nanoparticles (VNPs) genetically engineered to present osteogenic cues provide a promising method for biofunctionalizing hydrogels in bone tissue engineering. Flexible Potato virus X (PVX) nanoparticles substantially enhance the attachment and differentiation of human mesenchymal stem cells (hMSCs) by presenting the RGD motif, hydroxyapatite-binding peptide (HABP), or consecutive polyglutamates (E8) in a concentration-dependent manner. Therefore, it is hypothesized that Tobacco mosaic virus nanoparticles, which present 1.6 times more functional peptides than PVX, will meliorate such an impact. This study hypothesizes that cultivating hMSCs on a surface coated with a combination of two VNPs presenting peptides for either cell attachment or mineralization can achieve additionally enhancing effects on osteogenesis. Calcium minerals deposited by differentiating hMSCs increases two to threefold for this combination, while the Alkaline Phosphatase activity of hMSCs grown on the PVX-RGD/PVX-HABP-coated surface significantly surpasses any other VNP combination. Superior additive effects are observed for the first time by employing a combination of VNPs with varying functionalities. It is found that the flexible VNP geometry plays a more critical role than the concentration of functional peptides. In conclusion, various peptide-presenting plant VNPs exhibit an additive enhancing effect offering significant potential for effectively functionalizing cell-containing hydrogels in bone tissue engineering.

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.

Analysis of Engineered Tobacco Mosaic Virus and Potato Virus X Nanoparticles as Carriers for Biocatalysts.

Plant virus nanoparticles are promising candidates for the development of novel materials, including nanocomposites and scaffolds/carriers for functional molecules such as enzymes. Their advantages for enzyme immobilization include a modular organization, a robust and programmable structure, and a simple, cost-effective production. However, the activity of many enzymes relies on posttranslational modification and most plant viruses replicate in the cytoplasm, so functional enzymes cannot be displayed on the virus surface by direct coat protein fusions. An alternative display system to present the Trichoderma reesei endoglucanase Cel12A on potato virus X (PVX) using SpyTag/SpyCatcher (ST/SC) technology was recently developed by the authors, which allows the carrier and enzyme to be produced separately before isopeptide conjugation. Although kinetic analysis clearly indicated efficient biocatalyst activity, the PVX carrier interfered with substrate binding. To overcome this, the suitability of tobacco mosaic virus (TMV) was tested, which can also accommodate a larger number of ST peptides. We produced TMV particles displaying ST as a new platform for the immobilization of enzymes such as Cel12A, and compared its performance to the established PVX-ST platform in terms of catalytic efficiency. Although more enzyme molecules were immobilized on the TMV-ST particles, we found that the rigid scaffold and helical spacing significantly affected enzyme activity.

Targeted mutagenesis in Nicotiana tabacum ADF gene using shockwave-mediated ribonucleoprotein delivery increases osmotic stress tolerance.

DNA-free genome editing involves the direct introduction of ribonucleoprotein (RNP) complexes into cells, but this strategy has rarely been successful in plants. In the present study, we describe a new technique for the introduction of RNPs into plant cells involving the generation of cavitation bubbles using a pulsed laser. The resulting shockwave achieves the efficient transfection of walled cells in tissue explants by creating transient membrane pores. RNP-containing cells were rapidly identified by fluorescence microscopy, followed by regeneration and the screening of mutant plants by high-resolution melt analysis. We used this technique in Nicotiana tabacum to target the endogenous phytoene desaturase (PDS) and actin depolymerizing factor (ADF) genes. Genome-edited plants were produced with an efficiency of 35.2% for PDS and 16.5% for ADF. Further we evaluated the physiological, cellular and molecular effects of ADF mutations in T2 mutant plants under drought and salinity stress. The results suggest that ADF acts as a key regulator of osmotic stress tolerance in plants.

Three Alternative Treatment Protocols for the Efficient Inactivation of Potato Virus X.

Filamentous nanomaterials are flexible with a high aspect ratio, conferring unique mechanical, electromagnetic, and optical properties; promoting tissue penetration; and allowing the formation of hierarchical superstructures. The fabrication of synthetic nanofilaments with uniform properties is challenging, but this can be addressed by the use of filamentous plant viruses such as potato virus X (PVX), which are produced as monodisperse structures from a genetic template. To take advantage of PVX without risks to agriculture and the environment, it is necessary to inactivate the virus efficiently without disrupting its chemical and material properties. Herein, we report experiments showing that PVX can be completely inactivated by exposure to UV irradiation (0.5 J cm-2) or chemical treatment (1 mM ß-propiolactone or 10 mM formalin) without interfering with the chemical addressability of lysine or cysteine residues, which are typically used as conjugation handles for virus nanoparticle functionalization.

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.

Affinity of plant viral nanoparticle potato virus X (PVX) towards malignant B cells enables cancer drug delivery.

Non-Hodgkin's B cell lymphomas (NHL) include a diverse set of neoplasms that constitute ∼90% of all lymphomas and the largest subset of blood cancers. While chemotherapy is the first line of treatment, the efficacy of contemporary chemotherapies is hampered by dose-limiting toxicities. Partly due to suboptimal dosing, ∼40% of patients exhibit relapsed or refractory disease. Therefore more efficacious drug delivery systems are urgently needed to improve survival of NHL patients. In this study we demonstrate a new drug delivery platform for NHL based on the plant virus Potato virus X (PVX). We observed a binding affinity of PVX towards malignant B cells. In a metastatic mouse model of NHL, we show that systemically administered PVX home to tissues harboring malignant B cells. When loaded with the chemotherapy monomethyl auristatin (MMAE), the PVX nanocarrier enables effective delivery of MMAE to human B lymphoma cells in a NHL mouse model leading to inhibition of lymphoma growth in vivo and improved survival. Thus, PVX nanoparticle is a promising drug delivery platform for B cell malignancies.

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.

Presentation and Delivery of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand via Elongated Plant Viral Nanoparticle Enhances Antitumor Efficacy.

Potato virus X (PVX) is a flexuous plant virus-based nanotechnology with promise in cancer therapy. As a high aspect ratio biologic (13 × 515 nm), PVX has excellent spatial control in structures and functions, offering high-precision nanoengineering for multivalent display of functional moieties. Herein, we demonstrate the preparation of the PVX-based nanocarrier for delivery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a promising protein drug that induces apoptosis in cancer cells but not healthy cells. TRAIL bound to PVX by coordination bonds between nickel-coordinated nitrilotriacetic acid on PVX and His-tag on the protein could mimic the bioactive "membrane-bound" state in native TRAIL, resulting in an elongated nanoparticle displaying up 490 therapeutic protein molecules. Our data show that PVX-delivered TRAIL activates caspase-mediated apoptosis more efficiently compared to soluble TRAIL; also in vivo the therapeutic nanoparticle outperforms in delaying tumor growth in an athymic nude mouse model bearing human triple-negative breast cancer xenografts. This proof-of-concept work highlights the potential of filamentous plant virus nanotechnologies, particularly for targeting protein drug delivery for cancer therapy.

Biodistribution of Filamentous Plant Virus Nanoparticles: Pepino Mosaic Virus versus Potato Virus X.

Nanoparticles with high aspect ratios have favorable attributes for drug delivery and bioimaging applications based on their enhanced tissue penetration and tumor homing properties. Here, we investigated a novel filamentous viral nanoparticle (VNP) based on the Pepino mosaic virus (PepMV), a relative of the established platform Potato virus X (PVX). We studied the chemical reactivity of PepMV, produced fluorescent versions of PepMV and PVX, and then evaluated their biodistribution in mouse tumor models. We found that PepMV can be conjugated to various small chemical modifiers including fluorescent probes via the amine groups of surface-exposed lysine residues, yielding VNPs carrying payloads of up to 1600 modifiers per particle. Although PepMV and PVX share similarities in particle size and shape, PepMV achieved enhanced tumor homing and less nonspecific tissue distribution compared to PVX in mouse models of triple negative breast cancer and ovarian cancer. In conclusion, PepMV provides a novel tool for nanomedical research but more research is needed to fully exploit the potential of plant VNPs for health applications.

Biocatalytically induced surface modification of the tobacco mosaic virus and the bacteriophage M13.

Engineered viruses are finding an increasing number of applications in basic, translational research and materials science. Genetic and chemical engineering of capsids represents a key point for tailoring the properties of viral particles, but the synthetic efforts and limits accompanying these processes still hinder their usability. Here, a single-step highly selective biocatalytic functionalization approach is described, providing a general platform for virus-acrylate hybrid particles. The tobacco mosaic virus (TMV) and the bacteriophage M13 have been successfully modified via laccase induced free radical formation on the tyrosine residues through single electron oxidation as the initiating step and the free radicals subsequently react with acrylate-based monomers. This new approach can be extended to other biomolecular assemblies with surface exposed tyrosine residues, when the introduction of new functionalities is desired.

Nanovirus DNA-N encodes a protein mandatory for aphid transmission.

Nanoviruses possess a multipartite single-stranded DNA genome and are naturally transmitted to plants by various aphid species in a circulative non-propagative manner. Using the cloned genomic DNAs of faba bean necrotic stunt virus (FBNSV) for reconstituting nanovirus infections we analyzed the necessity of different virus components for infection and transmission by aphids. We found that in the absence of DNA-U1 and DNA-U2 symptom severity decreased, and in the absence of DNA-U1 the transmission efficiency decreased. Most significantly, we demonstrated that the protein encoded by DNA-N (NSP) is mandatory for aphid transmission. Moreover, we showed that the NSP of FBNSV could substitute for that of a distantly related nanovirus, pea necrotic yellow dwarf virus. Altering the FBNSV NSP by adding 13 amino acids to its carboxy-terminus resulted in an infectious but non-transmissible virus. We demonstrate that the NSP acts as a nanovirus transmission factor, the existence of which had been hypothesized earlier.

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.

Engineering Potato Virus X Particles for a Covalent Protein Based Attachment of Enzymes.

Plant virus nanoparticles are often used to display functional amino acids or small peptides, thus serving as building blocks in application areas as diverse as nanoelectronics, bioimaging, vaccination, drug delivery, and bone differentiation. This is most easily achieved by expressing coat protein fusions, but the assembly of the corresponding virus particles can be hampered by factors such as the fusion protein size, amino acid composition, and post-translational modifications. Size constraints can be overcome by using the Foot and mouth disease virus 2A sequence, but the compositional limitations cannot be avoided without the introduction of time-consuming chemical modifications. SpyTag/SpyCatcher technology is used in the present study to covalently attach the Trichoderma reesei endoglucanase Cel12A to Potato virus X (PVX) nanoparticles. The formation of PVX particles is confirmed by western blot, and the ability of the particles to display Cel12A is demonstrated by enzyme-linked immunosorbent assays and transmission electron microscopy. Enzymatic assays show optimal reaction conditions of 50 °C and pH 6.5, and an increased substrate conversion rate compared to free enzymes. It is concluded that PVX displaying the SpyTag can serve as new scaffold for protein display, most notably for proteins with post-translational modifications.

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.