Effect of the Coat Protein N-Terminal Domain Structure on the Structure and Physicochemical Properties of Virions of Potato Virus X and Alternanthera Mosaic Virus.

The amino acid sequences of the coat proteins (CPs) of the potexviruses potato virus X (PVX) and alternanthera mosaic virus (AltMV) share ~40% identity. The N-terminal domains of these proteins differ in the amino acid sequence and the presence of the N-terminal fragment of 28 residues (ΔN peptide) in the PVX CP. Here, we determined the effect of the N-terminal domain on the structure and physicochemical properties of PVX and AltMV virions. The circular dichroism spectra of these viruses differed significantly, and the melting point of PVX virions was 10-12°C higher than that of AltMV virions. Alignment of the existing high-resolution 3D structures of the potexviral CPs showed that the RMSD value between the Cα-atoms was the largest for the N-terminal domains of the two compared models. Based on the computer modeling, the ΔN peptide of the PVX CP is fully disordered. According to the synchrotron small-angle X-ray scattering (SAXS) data, the structure of CPs from the PVX and AltMV virions differ; in particular, the PVX CP has a larger portion of crystalline regions and, therefore, is more ordered. Based on the SAXS data, the diameters of the PVX and AltMV virions and helix parameters in solution were calculated. The influence of the conformation of the PVX CP N-terminal domain and its position relative to the virion surface on the virion structure was investigated. Presumably, an increased thermal stability of PVX virions vs. AltMV is provided by the extended N-terminal domain (ΔN peptide, 28 amino acid residues), which forms additional contacts between the adjacent CP subunits in the PVX virion.

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.

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.

Atomic structure of potato virus X, the prototype of the Alphaflexiviridae family.

Potato virus X (PVX) is a positive-sense single-stranded RNA (ssRNA) filamentous plant virus belonging to the Alphaflexiviridae family, considered in recent years as a tool for nanotechnology applications. We present the cryo-electron microscopy structure of the PVX particle at a resolution of 2.2 Å. The well-defined density of the coat proteins and of the genomic RNA allowed a detailed analysis of protein-RNA interactions, including those mediated by solvent molecules. The particle is formed by repeated segments made of 8.8 coat proteins, forming a left-handed helical structure. The RNA runs in an internal crevice along the virion, packaged in 5-nucleotide repeats in which the first four bases are stacked in the classical way, while the fifth is rotated and nearly perpendicular. The resolution of the structure described here suggests a mechanism for the virion assembly and potentially provides a platform for the rational design of antiviral compounds and for the use of PVX in nanotechnology.

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.

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.

Fabrication of Plant Virus-Based Thin Films to Modulate the Osteogenic Differentiation of Mesenchymal Stem Cells.

Stem cells can interact and respond to the extracellular nanoscale environment. Viral nanoparticles have been utilized as building blocks to control cell growth and differentiation. By integrating stem cell research and virus nanoparticle chemistry together, a systematic analysis of the effects of nanotopography on stem cell differentiation can be accomplished. The fabrication of thin films of the viral nanoparticles is particularly valuable for such studies. Here, we describe two methods to fabricate plant virus-based thin films and procedures to study the osteogenic differentiation of mesenchymal stem cells on plant virus-based substrates. The method makes use of wild-type tobacco mosaic virus (wt-TMV), RGD-modified TMV (TMV-RGD), turnip yellow mosaic virus (TYMV), cowpea mosaic virus (CPMV), turnip vein clearing virus (TVCV), and potato virus X (PVX) for development of bone tissue engineering biomaterials.

An isolate of Potato Virus X capsid protein from N. benthamiana: Insights from homology modeling and molecular dynamics simulation.

Since Potato Virus X (PVX) is easily transmitted mechanically between their hosts, its control is difficult. We have previously reported new isolate of this virus (PVX-Iran, GenBank Accession number FJ461343). However, the molecular basis of resistance breaking activity and its relation to capsid protein structure are still not well-understood. SDS-PAGE, ELISA, Western blot and RT-PCR molecular examinations were performed on the inoculated plants Nicotiana benthamiana. The pathological symptoms were related to the PVX isolate. The capsid protein (CP) structure were modeled based on homology and subjected to three independent 80 ns molecular dynamics minimization (GROMACS, OPLS force field) in the SPC water box. The RMSD, RMSF, SASA, and electrostatic properties were retrieved from the trajectories. Flexibility and hydrophilic nature of the N-terminal residues (1-34) of solvated CP could be observed in conformational changes upon minimization. The obtained structure was then docked with NbPCIP1 using ClusPro 2.0. The strong binding affinity of these two proteins (≈-16.0 Kcal mol-1) represents the formation of inclusion body and hence appearance of the symptoms.

Activation of innate immunity in primary human cells using a plant virus derived nanoparticle TLR7/8 agonist.

Rod-shaped virus-like nanoparticles (VLNP) made of papaya mosaic virus (PapMV) coat proteins (CP) self-assembled around a single stranded RNA (ssRNA) were showed to be a TLR7 agonist. Their utilization as an immune modulator in cancer immunotherapy was shown to be promising. To establish a clinical relevance in human for PapMV VLNP, we showed that stimulation of human peripheral blood mononuclear cells (PBMC) with VLNP induces the secretion of interferon alpha (IFNα) and other pro-inflammatory cytokines and chemokines. Plasmacytoid dendritic cells (pDCs) were activated and secreted IFN-α upon VLNP exposure. Monocyte-derived dendritic cells upregulate maturation markers and produce IL-6 in response to PapMV VLNP stimulation, which suggests the activation of TLR8. Finally, when co-cultured with NK cells, PapMV induced pDCs promoted the NK cytolytic activity against cancer cells. These data obtained with primary human immune cells further strengthen the clinical relevance of PapMV VLNPs as a cancer immunotherapy agent.

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.

A versatile papaya mosaic virus (PapMV) vaccine platform based on sortase-mediated antigen coupling.

BACKGROUND: Flexuous rod-shaped nanoparticles made of the coat protein (CP) of papaya mosaic virus (PapMV) have been shown to trigger innate immunity through engagement of toll-like receptor 7 (TLR7). PapMV nanoparticles can also serve as a vaccine platform as they can increase the immune response to fused peptide antigens. Although this approach shows great potential, fusion of antigens directly to the CP open reading frame (ORF) is challenging because the fused peptides can alter the structure of the CP and its capacity to self assemble into nanoparticles-a property essential for triggering an efficient immune response to the peptide. This represents a serious limitation to the utility of this approach as fusion of small peptides only is tolerated. RESULTS: We have developed a novel approach in which peptides are fused directly to pre-formed PapMV nanoparticles. This approach is based on the use of a bacterial transpeptidase (sortase A; SrtA) that can attach the peptide directly to the nanoparticle. An engineered PapMV CP harbouring the SrtA recognition motif allows efficient coupling. To refine our engineering, and to predict the efficacy of coupling with SrtA, we modeled the PapMV structure based on the known structure of PapMV CP and on recent reports revealing the structure of two closely related potexviruses: pepino mosaic virus (PepMV) and bamboo mosaic virus (BaMV). We show that SrtA can allow the attachment of long peptides [Influenza M2e peptide (26 amino acids) and the HIV-1 T20 peptide (39 amino acids)] to PapMV nanoparticles. Consistent with our PapMV structural model, we show that around 30% of PapMV CP subunits in each nanoparticle can be fused to the peptide antigen. As predicted, engineered nanoparticles were capable of inducing a strong antibody response to the fused antigen. Finally, in a challenge study with influenza virus, we show that mice vaccinated with PapMV-M2e are protected from infection. CONCLUSIONS: This technology will allow the development of vaccines harbouring long peptides containing several B and/or T cell epitopes that can contribute to a broad and robust protection from infection. The design can be fast, versatile and can be adapted to the development of vaccines for many infectious diseases as well as cancer vaccines.

Combination of Plant Virus Nanoparticle-Based in Situ Vaccination with Chemotherapy Potentiates Antitumor Response.

Immunotherapeutics are gaining more traction in the armamentarium used to combat cancer. Specifically, in situ vaccination strategies have gained interest because of their ability to alter the tumor microenvironment to an antitumor state. Herein, we investigate whether flexuous plant virus-based nanoparticles formed by the potato virus X (PVX) can be used as an immunotherapeutic for in situ vaccine monotherapy. We further developed dual chemo-immunotherapeutics by incorporating doxorubicin (DOX) into PVX yielding a dual-functional nanoparticle (PVX-DOX) or by coadministration of the two therapeutic regimes, PVX immunotherapy and DOX chemotherapy (PVX+DOX). In the context of B16F10 melanoma, PVX was able to elicit delayed tumor progression when administered as an intratumoral in situ vaccine. Furthermore, the coadministration of DOX via PVX+DOX enhanced the response of the PVX monotherapy through increased survival, which was also represented in the enhanced antitumor cytokine/chemokine profile stimulated by PVX+DOX when compared to PVX or DOX alone. Importantly, coadministered PVX+DOX was better for in situ vaccination than PVX loaded with DOX (PVX-DOX). Whereas the nanomedicine field strives to design multifunctional nanoparticles that integrate several functions and therapeutic regimens into a single nanoparticle, our data suggest a paradigm shift; some therapeutics may need to be administered separately to synergize and achieve the most potent therapeutic outcome. Altogether, our studies show that development of plant viral nanoparticles for in situ vaccines for treatment is a possibility, and dual mechanistic therapeutics can increase efficacy. Nonetheless, combining immunotherapeutics with cytolytic chemotherapy requires detailed investigation to inform optimal integration of cytolytic and immunotherapies and maximize synergy and efficacy.

Chemical addressability of potato virus X for its applications in bio/nanotechnology.

Potato virus X (PVX), a type member of the plant virus potexvirus group, offers a unique nanotechnology platform based on its high aspect ratio and flexible filamentous shape. The PVX platform has already been engineered and studied for its uses in imaging, drug delivery, and immunotherapies. While genetic engineering procedures are well established for PVX, there is limited information about chemical conjugation strategies for functionalizing PVX, partly due to the lack of structural information of PVX at high resolution. To overcome these challenges, we built a structural model of the PVX particle based on the available structures from pepino mosaic virus (PepMV), a close cousin of PVX. Using the model and a series of chemical conjugation experiments, we identified and probed the addressability of cysteine side chains. Chemical reactivity of cysteines was confirmed using Michael-addition and thiol-selective probes, including fluorescent dyes and biotin tags. LC/MS/MS was used to map Cys 121 as having the highest selectivity for modification. Finally, building on the availability of two reactive groups, the newly identified Cys and previously established Lys side chains, we prepared multifunctional PVX nanoparticles by conjugating Gd-DOTA for magnetic resonance imaging (MRI) to lysines and fluorescent dyes for optical imaging to cysteines. The resulting functionalized nanofilament could have applications in dual-modal optical-MRI imaging applications. These results further extend the understanding of the chemical properties of PVX and enable development of novel multifunctional platforms in bio/nanotechnology.

The cysteine residues at the C-terminal tail of Bamboo mosaic virus triple gene block protein 2 are critical for efficient plasmodesmata localization of protein 1 in the same block.

The movement of some plant viruses are accomplished by three proteins encoded by a triple gene block (TGB). The second protein (TGBp2) in the block is a transmembrane protein. This study was aimed to unravel the mechanism underlying the relatively inefficient cell-to-cell movement of Bamboo mosaic virus (BaMV) caused by amino acid substitutions for the three Cys residues, Cys-109, Cys-112 and Cys-119, at the C-terminal tail of TGBp2. Results from confocal microscopy revealed that substitutions of the three Cys residues of TGBp2, especially Cys-109 and Cys-112, would reduce the efficiency of TGBp2- and TGBp3-dependent PD localization of TGBp1. Moreover, there is an additive effect of the substitutions on reducing the efficiency of PD localization of TGBp1. These results indicate that the Cys residues in the C-terminal tail region of TGBp2 participate in the TGBp2- and TGBp3-dependent PD localization of TGBp1, and thus influence the cell-to-cell movement capability of BaMV.

Thermal conversion of filamentous potato virus X into spherical particles with different properties from virions.

We developed a method for the fast transformation of virions of tobacco mosaic virus (TMV) in so-called spherical particles (SPs) of different sizes. These SPs turned out to be highly useful for the preparation of different kinds of important biotechnological products. In this communication, we report that a representative of the flexuous helical virus group-potato virus X (PVX), produces SPs as well, but these SPs differ from TMV SPs in several important aspects. PVX SPs may be useful biotechnological devices.

Production of Immunoabsorbent Nanoparticles by Displaying Single-Domain Protein A on Potato Virus X.

The combination of antibodies with nanoparticles provides wide-ranging applications in biosensing. While several covalent presentation strategies have been established, there is need for alternative, non-covalent methods to provide a routine for scalable nanomanufacturing. We report the multivalent presentation of the B domain of Staphylococcus aureus protein A (SpAB) on potato virus X (PVX) nanoparticles. Three different synthetic strategies were used to obtain chimeric PVX(SpAB) filaments. The protein A fragments displayed on the surface of all three PVX chimeras remained fully functional as an immunoabsorbent for antibody capture enabling biosensing. The new biomaterials presented could find applications as diagnostic tools for biomedical or environmental monitoring.

Ultrastructural insights into tomato infections caused by three different pathotypes of Pepino mosaic virus and immunolocalization of viral coat proteins.

This paper presents studies on an ultrastructural analysis of plant tissue infected with different pathotypes of Pepino mosaic virus (PepMV) and the immunolocalization of viral coat proteins. Because the PepMV virus replicates with a high mutation rate and exhibits significant genetic diversity, therefore, isolates of PepMV display a wide range of symptoms on infected plants. In this work, tomato plants of the Beta Lux cultivar were inoculated mechanically with three pathotypes representing the Chilean 2 (CH2) genotype: mild (PepMV-P22), necrotic (PepMV-P19) and yellowing (PepMV-P5-IY). The presence of viral particles in all infected plants in the different compartments of various cell types (i.e. spongy and palisade mesophyll, sieve elements and xylem vessels) was revealed via ultrastructural analyses. For the first time, it was possible to demonstrate the presence of crystalline inclusions, composed of virus-like particles. In the later stage of PepMV infection (14 dpi) various pathotype-dependent changes in the structure of the individual organelles (i.e. mitochondria, chloroplasts) were found. The strongest immunogold labeling of the viral coat proteins was also observed in plants infected by necrotic isolates. A large number of viral coat proteins were marked in the plant conductive elements, both xylem and phloem.

In vitro and in vivo toxicity evaluation of plant virus nanocarriers.

The use of biological self-assembling materials, plant virus nanoparticles in particular, appears very intriguing as it allows a great choice of symmetries and dimensions, easy chemical and biological engineering of both surface and/or internal cavity as well as safe and rapid production in plants. In this perspective, we present an initial evaluation of the safety profile of two structurally different plant viruses produced in Nicotiana benthamiana L. plants: the filamentous Potato virus X and the icosahedral Tomato bushy stunt virus. In vitro haemolysis assay was used to test the cytotoxic effects, which could arise by pVNPs interaction with cellular membranes, while early embryo assay was used to evaluate toxicity and teratogenicity in vivo. Data indicates that these structurally robust particles, still able to infect plants after incubation in serum up to 24h, have neither toxic nor teratogenic effects in vitro and in vivo. This work represents the first safety-focused characterization of pVNPs in view of their possible use as drug delivery carriers.

Stealth filaments: Polymer chain length and conformation affect the in vivo fate of PEGylated potato virus X.

Nanoparticles hold great promise for delivering medical cargos to cancerous tissues to enhance contrast and sensitivity of imaging agents or to increase specificity and efficacy of therapeutics. A growing body of data suggests that nanoparticle shape, in combination with surface chemistry, affects their in vivo fates, with elongated filaments showing enhanced tumor targeting and tissue penetration, while promoting immune evasion. The synthesis of high aspect ratio filamentous materials at the nanoscale remains challenging using synthetic routes; therefore we turned toward nature's materials, developing and studying the filamentous structures formed by the plant virus potato virus X (PVX). We recently demonstrated that PVX shows enhanced tumor homing in various preclinical models. Like other nanoparticle systems, the proteinaceous platform is cleared from circulation and tissues by the mononuclear phagocyte system (MPS). To increase bioavailability we set out to develop PEGylated stealth filaments and evaluate the effects of PEG chain length and conformation on pharmacokinetics, biodistribution, as well as potential immune and inflammatory responses. We demonstrate that PEGylation effectively reduces immune recognition while increasing pharmacokinetic profiles. Stealth filaments show reduced interaction with cells of the MPS; the protein:polymer hybrids are cleared from the body tissues within hours to days indicating biodegradability and biocompatibility. Tissue compatibility is indicated with no apparent inflammatory signaling in vivo. Tailoring PEG chain length and conformation (brush vs. mushroom) allows tuning of the pharmacokinetics, yielding long-circulating stealth filaments for applications in nanomedicine.