Tomato Bushy Stunt Virus Nanoparticles as a Platform for Drug Delivery to Shh-Dependent Medulloblastoma

Medulloblastoma (MB) is a primary central nervous system tumor affecting mainly young children. New strategies of drug delivery are urgent to treat MB and, in particular, the SHH-dependent subtype—the most common in infants—in whom radiotherapy is precluded due to the severe neurological side effects. Plant virus nanoparticles (NPs) represent an innovative solution for this challenge. Tomato bushy stunt virus (TBSV) was functionally characterized as a carrier for drug targeted delivery to a murine model of Shh-MB. The TBSV NPs surface was genetically engineered with peptides for brain cancer cell targeting, and the modified particles were produced on a large scale using Nicotiana benthamiana plants. Tests on primary cultures of Shh-MB cells allowed us to define the most efficient peptides able to induce specific uptake of TBSV. Immunofluorescence and molecular dynamics simulations supported the hypothesis that the specific targeting of the NPs was mediated by the interaction of the peptides with their natural partners and reinforced by the presentation in association with the virus. In vitro experiments demonstrated that the delivery of Doxorubicin through the chimeric TBSV allowed reducing the dose of the chemotherapeutic agent necessary to induce a significant decrease in tumor cells viability. Moreover, the systemic administration of TBSV NPs in MB symptomatic mice, independently of sex, confirmed the ability of the virus to reach the tumor in a specific manner. A significant advantage in the recognition of the target appeared when TBSV NPs were functionalized with the CooP peptide. Overall, these results open new perspectives for the use of TBSV as a vehicle for the targeted delivery of chemotherapeutics to MB in order to reduce early and late toxicity.

Tomato Bushy Stunt Virus nanoparticles as a Platform for Drug Delivery to Shh-dependent Medulloblastoma

Background: Medulloblastoma (MB) is a primary central nervous system tumor that affects mainly young children. New strategies of drug delivery are urgent to treat this cancer and, in particular, the SHH-dependent subtype - the most common subgroup in infants - in whom radiotherapy is precluded due to the severe neurological side effects. Plant virus nanoparticles represent an innovative solution to approach this challenge.Methods: Tomato Bushy Stunt virus (TBSV) was functionally characterized using a murine model as carrier for targeted delivery to Shh-MB. The TBSV nanoparticles surface was engineered with peptides described as enabling targeting to brain cancer cells and the modified particles were then produced on large scale using Nicotiana benthamiana plants. Results: Tests on primary cultures of Shh-MB cells and on their cerebellar precursors allowed to define the most efficient peptides able to induce specific uptake of the viral NPs. Immunofluorescence and molecular dynamics simulations supported the hypothesis that the specific targeting of the NPs was mediated by the interaction of the peptides with their natural partners and reinforced by the presentation in association to the viral particle. In vitro experiments demonstrated that the delivery of Doxorubicin through the chimeric TBSV particles allowed to reduce the dose of the chemotherapeutic agent necessary to induce a significant decrease in tumor cells viability. Moreover, the systemic administration of TBSV nanoparticles in MB symptomatic mice confirmed the ability of the virus particles to reach the tumor in a specific manner. A significant advantage in the recognition of the target appeared when TBSV NPs were functionalized with the CooP peptide.Conclusion: Overall, these results open new perspectives for the use of TBSV particles as vehicle for the targeted delivery of chemotherapeutics to MB in order to reduce early and late toxicity.

Differential Response of Mycorrhizal Plants to Tomato bushy stunt virus and Tomato mosaic virus Infection

Tomato bushy stunt virus (TBSV) and Tomato mosaic virus (ToMV) are important economic pathogens in tomato fields. Rhizoglomus irregulare is a species of arbuscular mycorrhizal (AM) fungus that provides nutrients to host plants. To understand the effect of R. irregulare on the infection by TBSV/ToMV in tomato plants, in a completely randomized design, five treatments, including uninfected control plants without AM fungi (C), uninfected control plants with AM fungi (M) TBSV/ToMV-infected plants without AM fungi (V), TBSV/ToMV-infected plants before mycorrhiza (VM) inoculation, and inoculated plants with mycorrhiza before TBSV/ToMV infection (MV), were studied. Factors including viral RNA accumulation and expression of Pathogenesis Related proteins (PR) coding genes including PR1, PR2, and PR3 in the young leaves were measured. For TBSV, a lower level of virus accumulation and a higher expression of PR genes in MV plants were observed compared to V and VM plants. In contrast, for ToMV, a higher level of virus accumulation and a lower expression of PR genes in MV plants were observed as compared to V and VM plants. These results indicated that mycorrhizal symbiosis reduces or increases the viral accumulation possibly via the regulation of PR genes in tomato plants.

Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana

Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant–microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant–virus interactions in complement to Saccharomyces cerevisiae.