A Multifunctionalized Potyvirus-Derived Nanoparticle That Targets and Internalizes into Cancer Cells.

Plant viral nanoparticles (VNPs) are attractive to nanomedicine researchers because of their safety, ease of production, resistance, and straightforward functionalization. In this paper, we developed and successfully purified a VNP derived from turnip mosaic virus (TuMV), a well-known plant pathogen, that exhibits a high affinity for immunoglobulins G (IgG) thanks to its functionalization with the Z domain of staphylococcal Protein A via gene fusion. We selected cetuximab as a model IgG to demonstrate the versatility of this novel TuMV VNP by developing a fluorescent nanoplatform to mark tumoral cells from the Cal33 line of a tongue squamous cell carcinoma. Using confocal microscopy, we observed that fluorescent VNP-cetuximab bound selectively to Cal33 and was internalized, revealing the potential of this nanotool in cancer research.

Antigen-functionalized turnip mosaic virus nanoparticles increase antibody sensing in saliva. A case study with SARS-CoV-2 RBD.

Nanoparticles derived from plant viruses play an important role in nanomedicine due to their biocompatibility, self-assembly and easily-modifiable surface. In this study, we developed a novel platform for increasing antibody sensing using viral nanoparticles derived from turnip mosaic virus (TuMV) functionalized with SARS-CoV-2 receptor binding domain (RBD) through three different methods: chemical conjugation, gene fusion and the SpyTag/SpyCatcher technology. Even though gene fusion turned out to be unsuccessful, the other two constructs were proven to significantly increase antibody sensing when tested with saliva of patients with different infection and vaccination status to SARS-CoV-2. Our findings show the high potential of TuMV nanoparticles in the fields of diagnostics and immunodetection, being especially attractive for the development of novel antibody sensing devices.

Intrinsic disorder in the dynamic evolution of structure, stability, and flexibility of potyviral VLP assemblies: A computational study.

An all-atom Molecular Dynamics (MD) study was applied to three viral nanoparticles (VLPs) of Turnip mosaic virus (TuMV), a potyvirus: the particles genetically functionalized with two peptides, VIP (human vasoactive intestinal peptide) and VEGFR (peptide derived from the human receptor 3 of the vascular endothelial growth factor), and the non-functionalized VLP. Previous experimental results showed that VIP-VLP was the only construct of the three that was not viable. VLPs subjected to our MD study were modeled by four complete turns of the particle involving 35 subunits of the coat protein (CP). The MD simulations showed differences in structures and interaction energies associated to the crucial contribution of the disordered N-terminal arms of CP to the global stability of the particle. These differences suggested an overall stability greater in VEGFR-VLP and smaller in VIP-VLP as compared to the unfunctionalized VLP. Our novel MD study of potyviral VLPs revealed essential clues about structure and interactions of these assembled protein particles and suggests that the computational prediction of the viability of VLPs can be a valuable contribution in the field of viral nanobiotechnology.

Structural intrinsic disorder in a functionalized potyviral coat protein as a main viability determinant of its assembled nanoparticles.

The viability of viral-derived nanoparticles (virions and VLPs) aimed to nanobiotechnological functionalizations of the coat protein (CP) of turnip mosaic virus has been studied by means of advanced computational methodologies that include molecular dynamics. The study has allowed to model the structure of the complete CP and its functionalization with three different peptides and obtain essential structural features such as order/disorder, interactions, and electrostatic potentials of their constituent domains. The results provide for the first time a dynamic view of a complete potyvirus CP, since experimental available structures so far obtained lack N- and C-terminal segments. The relevance of disorder in the most distal N-terminal subdomain, and the interaction of the less distal N-terminal subdomain with the highly ordered CP core, stand out as crucial characteristic for a viable CP. Preserving them proved of outmost importance to obtain viable potyviral CPs presenting peptides at their N-terminus.

Corrigendum: Suitability of potyviral recombinant virus-like particles bearing a complete food allergen for immunotherapy vaccines.

[This corrects the article DOI: 10.3389/fimmu.2022.986823.].

Isopeptide Bonding In Planta Allows Functionalization of Elongated Flexuous Proteinaceous Viral Nanoparticles, including Non-Viable Constructs by Other Means.

Plant viral nanoparticles (VNPs) have become an attractive platform for the development of novel nanotools in the last years because of their safety, inexpensive production, and straightforward functionalization. Turnip mosaic virus (TuMV) is one example of a plant-based VNP used as a nanobiotechnological platform either as virions or as virus-like particles (VLPs). Their functionalization mainly consists of coating their surface with the molecules of interest via chemical conjugation or genetic fusion. However, because of their limitations, these two methods sometimes result in non-viable constructs. In this paper, we applied the SpyTag/SpyCatcher technology as an alternative for the functionalization of TuMV VLPs with peptides and proteins. We chose as molecules of interest the green fluorescent protein (GFP) because of its good traceability, as well as the vasoactive intestinal peptide (VIP), given the previous unsuccessful attempts to functionalize TuMV VNPs by other methods. The successful conjugation of VLPs to GFP and VIP using SpyTag/SpyCatcher was confirmed through Western blot and electron microscopy. Moreover, the isopeptide bond between SpyTag and SpyCatcher occurred in vivo in co-agroinfiltrated Nicotiana benthamiana plants. These results demonstrated that SpyTag/SpyCatcher improves TuMV functionalization compared with previous approaches, thus implying the expansion of the application of the technology to elongated flexuous VNPs.

Suitability of potyviral recombinant virus-like particles bearing a complete food allergen for immunotherapy vaccines.

Virus-like particles (VLPs) have been gaining attention as potential platforms for delivery of cargos in nanomedicine. Although animal viruses are largely selected due to their immunostimulatory capacities, VLPs from plant viruses constitute a promising alternative to be considered. VLPs derived from Turnip mosaic virus (TuMV) have proven to present a tridimensional structure suited to display molecules of interest on their surface, making them interesting tools to be studied in theragnostic strategies. Here, we study their potential in the treatment of food allergy by genetically coupling TuMV-derived VLPs to Pru p 3, one of the most dominant allergens in Mediterranean climates. VLPs-Pru p 3 were generated by cloning a synthetic gene encoding the TuMV coat protein and Pru p 3, separated by a linker, into a transient high-expression vector, followed by agroinfiltration in Nicotiana benthamiana plants. The generated fusion protein self-assembled in planta to form the VLPs, which were purified by exclusion chromatography. Their elongated morphology was confirmed by electron microscopy and their size (~400 nm), and monodispersity was confirmed by dynamic light scattering. Initial in vitro characterization confirmed that they were able to induce proliferation of human immune cells. This proliferative capability was enhanced when coupled with the natural lipid ligand of Pru p 3. The resultant formulation, called VLP-Complex, was also able to be transported by intestinal epithelial cells, without affecting the monolayer integrity. In light of all these results, VLP-Complex was furtherly tested in a mouse model of food allergy. Sublingual administration of VLP-Complex could effectively reduce some serological markers associated with allergic responses in mice, such as anti-Pru p 3 sIgE and sIgG2a. Noteworthy, no associated macroscopic, nephritic, or hepatic toxicity was detected, as assessed by weight, blood urea nitrogen (BUN) and galectin-3 analyses, respectively. Our results highlight the standardized production of allergen-coated TuMV-VLPs in N. benthamiana plants. The resulting formula exerts notable immunomodulatory properties without the need for potentially hazardous adjuvants. Accordingly, no detectable toxicity associated to their administration was detected. As a result, we propose them as good candidates to be furtherly studied in the treatment of immune-based pathologies.

Gelatin-methacryloyl hydrogels containing turnip mosaic virus for fabrication of nanostructured materials for tissue engineering.

Current tissue engineering techniques frequently rely on hydrogels to support cell growth, as these materials strongly mimic the extracellular matrix. However, hydrogels often need ad hoc customization to generate specific tissue constructs. One popular strategy for hydrogel functionalization is to add nanoparticles to them. Here, we present a plant viral nanoparticle the turnip mosaic virus (TuMV), as a promising additive for gelatin methacryloyl (GelMA) hydrogels for the engineering of mammalian tissues. TuMV is a flexuous, elongated, tubular protein nanoparticle (700-750 nm long and 12-15 nm wide) and is incapable of infecting mammalian cells. These flexuous nanoparticles spontaneously form entangled nanomeshes in aqueous environments, and we hypothesized that this nanomesh structure could serve as a nanoscaffold for cells. Human fibroblasts loaded into GelMA-TuMV hydrogels exhibited similar metabolic activity to that of cells loaded in pristine GelMA hydrogels. However, cells cultured in GelMA-TuMV formed clusters and assumed an elongated morphology in contrast to the homogeneous and confluent cultures seen on GelMA surfaces, suggesting that the nanoscaffold material per se did not favor cell adhesion. We also covalently conjugated TuMV particles with epidermal growth factor (EGF) using a straightforward reaction scheme based on a Staudinger reaction. BJ cells cultured on the functionalized scaffolds increased their confluency by approximately 30% compared to growth with unconjugated EGF. We also provide examples of the use of GelMA-TuMV hydrogels in different biofabrication scenarios, include casting, flow-based-manufacture of filaments, and bioprinting. We envision TuMV as a versatile nanobiomaterial that can be useful for tissue engineering.

Antitumor applications of polyphenol-conjugated turnip mosaic virus-derived nanoparticles.

Background: Filamentous plant virus-derived nanoparticles are biodegradable and noninfectious to humans. Their structure is also amenable to chemical modifications. They constitute an appealing material for biomedical applications including imaging and drug delivery. We had previously used turnip mosaic virus-derived nanoparticles (TuMV-NPs) to increase antibody-sensing in vivo, to prevent biofilm formation and to build biological nanoscaffolds. Materials & methods: We analyzed TuMV-NP biodistribution and tumor homing using in vivo imaging. We studied in vitro the interaction with human cancer cell lines and the antiproliferative effect of epigallocatechin gallate-functionalized TuMV-NPs. Results & conclusion: TuMV-NPs are efficiently internalized by human cells and show good tumor homing. The antiproliferative effect of epigallocatechin gallate-TuMV-NPs suggests that they could offer a potential anticancer therapy.

Cancer is the second leading cause of death worldwide, just behind cardiovascular disease. It accounts for nearly 10 million deaths annually, and new strategies to improve early detection and drug delivery are urgently needed. Nanoparticles are small structures within the nanometer range (1 billionth of a meter) that can be used to deliver either an imaging probe (tracer) to allow the detection of a tumor or drugs to kill tumor cells. There are many types of nanoparticles; those based on plant viruses are especially appealing for biomedical purposes because they are biodegradable and noninfectious to humans. Also, their physicochemical properties, such as symmetry, uniformity and loading capacity, make them excellent nanocarriers. We report here for the first time the ability of nanoparticles derived from the turnip mosaic virus (TuMV), a well-known virus naturally infecting cruciferous plants (e.g., broccoli, turnip, radish, cabbage) but not humans, to deliver a fluorescent imaging probe that allows tumor detection in vivo. Moreover, TuMV nanoparticles were used to deliver a natural chemotherapeutic agent of plant origin to different types of tumor cells (lung, colorectal, breast, and head and neck), showing increased antiproliferative capacity compared to the nonvehiculized drug.

The Effects of Turnip Mosaic Virus Infections on the Deposition of Secondary Cell Walls and Developmental Defects in Arabidopsis Plants Are Virus-Strain Specific.

Two isolates of Turnip mosaic virus (UK 1 and JPN 1), representative of two different viral strains, induced differential alterations on secondary cell wall (SCW) development in Arabidopsis thaliana, suggesting cell-type specific effects of these viral infections. These potential effects were analyzed in inflorescence stems and flowers of infected plants, together with other possible cellular effects of the infections. Results obtained from macroscopic and histochemical analyses showed that infection with either virus significantly narrowed stem area, but defects in SCW were only found in JPN 1 infections. In flowers, reduced endothecium lignification was also found for JPN 1, while UK 1 infections induced severe floral cell and organ development alterations. A transcriptomic analysis focused on genes controlling and regulating SCW formation also showed notable differences between both viral isolates. UK 1 infections induced a general transcriptional decrease of most regulatory genes, whereas a more complex pattern of alterations was found in JPN 1 infections. The role of the previously identified viral determinant of most developmental alterations, the P3 protein, was also studied through the use of viral chimeras. No SCW alterations or creeping habit growth were found in infections by the chimeras, indicating that if the P3 viral protein is involved in the determination of these symptoms, it is not the only determinant. Finally, considerations as to the possibility of a taxonomical reappraisal of these TuMV viral strains are provided.

The C-Terminal Half of SARS-CoV-2 Nucleocapsid Protein, Industrially Produced in Plants, Is Valid as Antigen in COVID-19 Serological Tests.

BACKGROUND: The fight against the current coronavirus disease 2019 (COVID-19) pandemic has created a huge demand of biotechnological, pharmaceutical, research and sanitary materials at unprecedented scales. One of the most urgent demands affects the diagnostic tests. The growing need for rapid and accurate laboratory diagnostic tests requires the development of biotechnological processes aimed at producing reagents able to cope with this demand in a scalable, cost-effective manner, with rapid turnaround times. This is particularly applicable to the antigens employed in serological tests. Recombinant protein expression using plants as biofactories is particularly suitable for mass production of protein antigens useful in serological diagnosis, with a neat advantage in economic terms. METHODS: We expressed a large portion of the nucleoprotein (N) derived from SARS-CoV-2 in Nicotiana benthamiana plants. After purification, the recombinant N protein obtained was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to SARS-CoV-2 in human sera. To validate the ELISA, a panel of 416 sera from exposed personnel at essential services in Madrid City Council were tested, and the results compared to those obtained by another ELISA, already validated, used as reference. Furthermore, a subset of samples for which RT-PCR results were available were used to confirm sensitivity and specificity of the test. RESULTS: The performance of the N protein expressed in plants as antigen in serologic test for SARS-CoV-2 antibody detection was shown to be highly satisfactory, with calculated diagnostic sensitivity of 96.41% (95% CI: 93.05-98.44) and diagnostic specificity of 96.37 (95% CI: 93.05-98.44) as compared to the reference ELISA, with a kappa (K) value of 0.928 (95% CI:0.892-0.964). Furthermore, the ELISA developed with plant-derived N antigen detected SARS-CoV-2 antibodies in 84 out of 93 sera from individuals showing RT-PCR positive results (86/93 for the reference ELISA). CONCLUSION: This study demonstrates that the N protein part derived from SARS-CoV-2 expressed in plants performs as a perfectly valid antigen for use in COVID-19 diagnosis. Furthermore, our results support the use of this plant platform for expression of recombinant proteins as reagents for COVID-19 diagnosis. This platform stands out as a convenient and advantageous production system, fit-for-purpose to cope with the current demand of this type of biologicals in a cost-effective manner, making diagnostic kits more affordable.

High overexpression of CERES, a plant regulator of translation, induces different phenotypical defence responses during TuMV infection.

Mutations in the eukaryotic translation initiation factors eIF4E and eIF(iso)4E confer potyvirus resistance in a range of plant hosts. This supports the notion that, in addition to their role in translation of cellular mRNAs, eIF4E isoforms are also essential for the potyvirus cycle. CERES is a plant eIF4E- and eIF(iso)4E-binding protein that, through its binding to the eIF4Es, modulates translation initiation; however, its possible role in potyvirus resistance is unknown. In this article, we analyse if the ectopic expression of AtCERES is able to interfere with turnip mosaic virus replication in plants. Our results demonstrate that, during infection, the ectopic expression of CERES in Nicotiana benthamiana promotes the development of a mosaic phenotype when it is accumulated to moderate levels, but induces veinal necrosis when it is accumulated to higher levels. This necrotic process resembles a hypersensitive response (HR)-like response that occurs with different HR hallmarks. Remarkably, Arabidopsis plants inoculated with a virus clone that promotes high expression of CERES do not show signs of infection. These final phenotypical outcomes are independent of the capacity of CERES to bind to eIF4E. All these data suggest that CERES, most likely due to its leucine-rich repeat nature, could act as a resistance protein, able to promote a range of different defence responses when it is highly overexpressed from viral constructs.

Biofabrication of muscle fibers enhanced with plant viral nanoparticles using surface chaotic flows.

Multiple human tissues exhibit fibrous nature. Therefore, the fabrication of hydrogel filaments for tissue engineering is a trending topic. Current tissue models are made of materials that often require further enhancement for appropriate cell attachment, proliferation and differentiation. Here we present a simple strategy, based on the use of surface chaotic flows amenable to mathematical modeling, to fabricate continuous, long and thin filaments of gelatin methacryloyl (GelMA). The fabrication of these filaments is achieved by chaotic advection in a finely controlled and miniaturized version of the journal bearing system. A drop of GelMA pregel is injected on a higher-density viscous fluid (glycerin) and a chaotic flow is applied through an iterative process. The millimeter-scale hydrogel drop is exponentially deformed and elongated to generate a meter-scale fiber, which was then polymerized under UV-light exposure. Computational fluid dynamic (CFD) simulations are conducted to determine the characteristics of the flow and design the experimental conditions for fabrication of the fibers. GelMA fibers were effectively used as scaffolds for C2C12 myoblast cells. Experimental results demonstrate an accurate accordance with CFD simulations for the predicted length of the fibers. Plant-based viral nanoparticles (i.e.Turnip mosaic virus; TuMV) were then integrated to the hydrogel fibers as a secondary nano-scaffold for cells for enhanced muscle tissue engineering. The addition of TuMV significantly increased the metabolic activity of the cell-seeded fibers (p* < 0.05), strengthened cell attachment throughout the first 28 d, improved cell alignment, and promoted the generation of structures that resemble natural mammal muscle tissues. Chaotic two-dimensional-printing is proven to be a viable method for the fabrication of hydrogel fibers. The combined use of thin and long GelMA hydrogel fibers enhanced with flexuous virions offers a promising alternative for scaffolding of muscle cells and show potential to be used as cost-effective models for muscle tissue engineering purposes.

Association between flower stalk elongation, an Arabidopsis developmental trait, and the subcellular location and movement dynamics of the nonstructural protein P3 of Turnip mosaic virus.

Virus infections affect plant developmental traits but this aspect of the interaction has not been extensively studied so far. Two strains of Turnip mosaic virus differentially affect Arabidopsis development, especially flower stalk elongation, which allowed phenotypical, cellular, and molecular characterization of the viral determinant, the P3 protein. Transiently expressed wild-type green fluorescent protein-tagged P3 proteins of both strains and selected mutants of them revealed important differences in their behaviour as endoplasmic reticulum (ER)-associated peripheral proteins flowing along the reticulum, forming punctate accumulations. Three-dimensional (3D) model structures of all expressed P3 proteins were computationally constructed through I-TASSER protein structure predictions, which were used to compute protein surfaces and map electrostatic potentials to characterize the effect of amino acid changes on features related to protein interactions and to phenotypical and subcellular results. The amino acid at position 279 was the main determinant affecting stalk development. It also determined the speed of ER-flow of the expressed proteins and their final location. A marked change in the protein surface electrostatic potential correlated with changes in subcellular location. One single amino acid in the P3 viral protein determines all the analysed differential characteristics between strains differentially affecting flower stalk development. A model proposing a role of the protein in the intracellular movement of the viral replication complex, in association with the viral 6K2 protein, is proposed. The type of association between both viral proteins could differ between the strains.

Turnip Mosaic Virus Coat Protein Deletion Mutants Allow Defining Dispensable Protein Domains for 'in Planta' eVLP Formation.

The involvement of different structural domains of the coat protein (CP) of turnip mosaic virus, a potyvirus, in establishing and/or maintaining particle assembly was analyzed through deletion mutants of the protein. In order to identify exclusively those domains involved in protein-protein interactions within the particle, the analysis was performed by agroinfiltration "in planta", followed by the assessment of CP accumulation in leaves and the assembly of virus-like particles lacking nucleic acids, also known as empty virus-like particles (eVLP). Thus, the interactions involving viral RNA could be excluded. It was found that deletions precluding eVLP assembly did not allow for protein accumulation either, probably indicating that non-assembled CP protein was degraded in the plant leaves. Deletions involving the CP structural core were incompatible with particle assembly. On the N-terminal domain, only the deletion avoiding the subdomain involved in interactions with other CP subunits was incorporated into eVLPs. The C-terminal domain was shown to be more permissive to deletions. Assembled eVLPs were found for mutants, eliminating the whole domain. The C-terminal domain mutants were unusually long, suggesting some role of the domain in the regulation of particle length. The identification of the CP domains responsible for eVLP formation will allow for new approaches to protein stretch replacement with peptides or proteins of nanobiotechnological interest. Finally, specific cases of application are considered.

Polyphenol-Functionalized Plant Viral-Derived Nanoparticles Exhibit Strong Antimicrobial and Antibiofilm Formation Activities.

(-)-Epigallocatechin gallate (EGCG) is a polyphenolic compound that shows a number of health-promoting effects, especially a broad antimicrobial activity. Virus-derived nanoparticles (VNPs) represent a promising drug carrier since they possess properties like biodegradability and their surface and interior are highly modifiable. Turnip mosaic virus (TuMV) VNPs offer an attractive number of conjugation sites on the external surface. EGCG-TuMV VNPs were synthesized by Mannich condensation, and their antimicrobial activities against the model bacteria Sarcina lutea, Pseudomonas aeruginosa, and Dickeya dadantii were tested. EGCG-TuMV VNPs did not only maintain TuMV structure but also showed an enhanced antimicrobial activity over that found with free EGCG for all of the bacteria tested. Biofilm formation by P. aeruginosa was also inhibited by EGCG-TuMV VNPs, contrary to free EGCG, which induced higher amounts of biofilm mass in a concentration-dependent manner. Taken together, our results open highly promising perspectives for the antimicrobial exploitation of EGCG-TuMV VNPs.

Elongated Flexuous Plant Virus-Derived Nanoparticles Functionalized for Autoantibody Detection.

Nanoparticles derived from the elongated flexuous capsids of Turnip mosaic virus (TuMV) have been shown to be efficient tools for antibody sensing with a very high sensitivity if adequately functionalized with the corresponding epitopes. Taking advantage of this possibility, TuMV virus-like particles (VLPs) have been genetically derivatized with a peptide from the chaperonin Hsp60, a protein described to be involved in inflammation processes and autoimmune diseases. Antibodies against the peptide have been previously shown to have a diagnostic value in at least one autoimmune disease, multiple sclerosis. The functionalized Hsp60-VLPs showed their significant increase in sensing potency when compared to monoclonal antibody detection of the peptide in a conventional immunoassay. Additionally, the developed Hsp60-VLPs allowed the detection of autoantibodies against the Hsp60 peptide in an in vivo mouse model of dextran sodium sulfate (DSS)-induced colitis. The detection of minute amounts of the autoantibodies allowed us to perform the analysis of their evolution during the progression of the disease. The anti-Hsp60 autoantibody levels in the sera of the inflamed mice went down during the induction phase of the disease. Increased levels of the anti-HSP60 autoantibodies were detected during the resolution phase of the disease. An extension of a previously proposed model for the involvement of Hsp60 in inflammatory processes is considered, incorporating a role for Hsp60 autoantibodies. This, and related models, can now be experimentally tested thanks to the autoantibody detection hypersensitivity provided by the functionalized VLPs.

Structure of Turnip mosaic virus and its viral-like particles.

Turnip mosaic virus (TuMV), a potyvirus, is a flexible filamentous plant virus that displays a helical arrangement of coat protein copies (CPs) bound to the ssRNA genome. TuMV is a bona fide representative of the Potyvirus genus, one of most abundant groups of plant viruses, which displays a very wide host range. We have studied by cryoEM the structure of TuMV virions and its viral-like particles (VLPs) to explore the role of the interactions between proteins and RNA in the assembly of the virions. The results show that the CP-RNA interaction is needed for the correct orientation of the CP N-terminal arm, a region that plays as a molecular staple between CP subunits in the fully assembled virion.