Pharmacokinetics and efficacy of doxorubicin-loaded plant virus nanoparticles in preclinical models of cancer.

AIM: To compare the pharmacokinetics and efficacy of doxorubicin containing plant virus nanoparticles (PVNs) with PEGylated liposomal doxorubicin (PLD) and small molecule doxorubicin in two mouse models of cancer. MATERIALS & METHODS: Studies were performed in A375 melanoma and intraperitoneal SKOV3ip1 ovarian cancer xenografts. The PVNs were administered in lower and more frequent doses in the ovarian model. RESULTS: The PVNs were more efficacious than PLD and small molecule doxorubicin in the ovarian cancer model, but not in the melanoma cancer model. The pharmacokinetics profiles of the PVNs showed fast plasma clearance, but more efficient tumor delivery as compared with other carrier-mediated agents. CONCLUSION: PVNs administered at lower repeated doses provide both pharmacologic and efficacy advantages compared with PLD.

Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by viruses.

Five viruses were previously discovered infecting soybean cyst nematodes (SCN; Heterodera glycines) from greenhouse cultures maintained in Illinois. In this study, the five viruses [ScNV, ScPV, ScRV, ScTV, and SbCNV-5] were detected within SCN greenhouse and field populations from North Carolina (NC) and Missouri (MO). The prevalence and titers of viruses in SCN from 43 greenhouse cultures and 25 field populations were analyzed using qRT-PCR. Viral titers within SCN greenhouse cultures were similar throughout juvenile development, and the presence of viral anti-genomic RNAs within egg, second-stage juvenile (J2), and pooled J3 and J4 stages suggests active viral replication within the nematode. Viruses were found at similar or lower levels within field populations of SCN compared with greenhouse cultures of North Carolina populations. Five greenhouse cultures harbored all five known viruses whereas in most populations a mixture of fewer viruses was detected. In contrast, three greenhouse cultures of similar descent to one another did not possess any detectable viruses and primarily differed in location of the cultures (NC versus MO). Several of these SCN viruses were also detected in Heterodera trifolii (clover cyst) and Heterodera schachtii (beet cyst), but not the other cyst, root-knot, or reniform nematode species tested. Viruses were not detected within soybean host plant tissue. If nematode infection with viruses is truly more common than first considered, the potential influence on nematode biology, pathogenicity, ecology, and control warrants continued investigation.

Development of abamectin loaded plant virus nanoparticles for efficacious plant parasitic nematode control.

Plant parasitic nematodes are one of the world's major agricultural pests, causing in excess of $157 billion in worldwide crop damage annually. Abamectin (Abm) is a biological pesticide with a strong activity against a wide variety of plant parasitic nematodes. However, Abm's poor mobility in the soil compromises its nematicide performance because of the limited zone of protection surrounding the growing root system of the plant. In this study, we manipulated Abm's soil physical chemistry by encapsulating Abm within the Red clover necrotic mosaic virus (RCNMV) to produce a plant virus nanoparticle (PVN) delivery system for Abm. The transmission electron microscopic and dynamic light scattering characterization of Abm-loaded PVN (PVN(Abm)) indicated the resultant viral capsid integrity and morphology comparable to native RCNMV. In addition, the PVN(Abm) significantly increased Abm's soil mobility while enabling a controlled release strategy for Abm's bioavailability to nematodes. As a result, PVN(Abm) enlarged the zone of protection from Meloidogyne hapla root knot nematodes in the soil as compared to treating with free Abm molecules. Tomato seedlings treated with PVN(Abm) had healthier root growth and a reduction in root galling demonstrating the success of this delivery system for the increased efficacy of Abm to control nematode damage in crops.

Nicotiana benthamiana: Its History and Future as a Model for Plant-Pathogen Interactions.

Nicotiana benthamiana is the most widely used experimental host in plant virology, due mainly to the large number of diverse plant viruses that can successfully infect it. Addi- tionally, N. benthamiana is susceptible to a wide variety of other plant-pathogenic agents (such as bacteria, oomycetes, fungi, and so on), making this species a cornerstone of host-pathogen research, particularly in the context of innate immunity and defense signaling. Moreover, because it can be genetically transformed and regenerated with good efficiency and is amenable to facile methods for virus- induced gene silencing or transient protein expression, N. benthamiana is rapidly gaining popularity in plant biology, particularly in studies requiring protein localization, inter- action, or plant-based systems for protein expression and purification. Paradoxically, despite being an indispensable research model, little is known about the origins, genetic variation, or ecology of the N. benthamiana accessions cur- rently used by the research community. In addition to ad- dressing these latter topics, the purpose of this review is to provide information regarding sources for tools and reagents that can be used to support research in N. benthamiana. Finally, we propose that N. benthamiana is well situated to become a premier plant cell biology model, particularly for the virology community, who as a group were the first to recognize the potential of this unique Australian native.

Loading and release mechanism of red clover necrotic mosaic virus derived plant viral nanoparticles for drug delivery of doxorubicin.

Loading and release mechanisms of Red clover necrotic mosaicvirus (RCNMV) derived plant viral nanoparticle (PVN) are shown for controlled delivery of the anticancer drug, doxorubicin (Dox). Previous studies demonstrate that RCNMV's structure and unique response to divalent cation depletion and re-addition enables Dox infusion to the viral capsid through a pore formation mechanism. However, by controlling the net charge of RCNMV outer surface and accessibility of RCNMV interior cavity, tunable release of PVN is possible via manipulation of the Dox loading capacity and binding locations (external surface-binding or internal capsid-encapsulation) with the RCNMV capsid. Bimodal release kinetics is achieved via a rapid release of surface-Dox followed by a slow release of encapsulated Dox. Moreover, the rate of Dox release and the amount of released Dox increases with an increase in environmental pH or a decrease in concentration of divalent cations. This pH-responsive Dox release from PVN is controlled by Fickian diffusion kinetics where the release rate is dependent on the location of the bound or loaded active molecule. In summary, controllable release of Dox-loaded PVNs is imparted by 1) formulation conditions and 2) driven by the capsid's pH- and ion- responsive functions in a given environment.

Polymeric systems incorporating plant viral nanoparticles for tailored release of therapeutics.

Therapeutic polylactide (PLA) nanofibrous matrices are fabricated by incorporating plant viral nanoparticles (PVNs) infused with fluorescent agents ethidium bromide (EtBr) and rhodamine (Rho), and cancer therapeutic doxorubicin (Dox). The native virus, Red clover necrotic mosaic virus (RCNMV), reversibly opens and closes upon exposure to the appropriate environmental stimuli. Infusing RCNMV with small molecules allows the incorporation of PVN(Active) into fibrous matrices via two methods: direct processing by in situ electrospinning of a polymer and PVNs solution or immersion of the matrix into a viral nanoparticle solution. Five organic solvents commonly in-use for electrospinning are evaluated for potential negative impact on RCNMV stability. In addition, leakage of rhodamine from the corresponding PVN(Rho) upon solvent exposure is determined. Incorporation of the PVN into the matrices are evaluated via transmission electron, scanning electron and fluorescent microscopies. Finally, the percent cumulative release of doxorubicin from both PLA nanofibers and PLA and polyethylene oxide (PEO) hybrid nanofibers demonstrate tailored release due to the incorporation of PVN(Dox) as compared to the control nanofibers with free Dox. Preliminary kinetic analysis results suggest a two-phase release profile with the first phase following a hindered Fickian transport mechanism for the release of Dox for the polymer-embedded PVNs. In contrast, the nanofiber matrices that incorporate PVNs through the immersion processing method followed a pseudo-first order kinetic transport mechanism.

The Red clover necrotic mosaic virus capsid protein N-terminal lysine-rich motif is a determinant of symptomatology and virion accumulation.

The interaction between viral capsid protein (CP) and its cognate viral RNA modulates many steps in the virus infection cycle, such as replication, translation and assembly. The N-terminal 50 amino acids of the Red clover necrotic mosaic virus (RCNMV) CP are rich in basic residues (especially lysine) and are essential for the core functions of the CP, namely RNA binding and virion assembly. To further elucidate additional biological roles for these basic residues, a series of alanine substitution mutations was introduced into infectious clones of RCNMV RNA-1 and assayed for symptomatology, virion formation and systemic infection. Infectivity assays conducted in Nicotiana benthamiana revealed that all nine alanine substitution mutants (ASMs) were competent for systemic infection. Two ASMs (K4A and K7A/K8A) induced severe symptoms and delayed the systemic spread of viral genomes when compared with wild-type RCNMV. However, these ASMs were still competent for virion formation. Three other ASMs (K25A, K33A and K38A) displayed milder symptoms and significant reductions in virion accumulation when compared with wild-type RCNMV, but retained the ability to spread systemically. Evidence from these last three ASMs, as well as a CP null mutant, showed that RCNMV is able to move systemically in N. benthamiana as a nonvirion form. These observations reaffirm the necessity of the N-terminal lysine-rich residues of the RCNMV CP for efficient virion accumulation. They also reveal additional roles for the CP in the modulation of host symptomatology, independent of its role in virion assembly and the rate of systemic viral movement in N. benthamiana.

Pectobacterium carotovorum elicits plant cell death with DspE/F but the P. carotovorum DspE does not suppress callose or induce expression of plant genes early in plant-microbe interactions.

The broad-host-range bacterial soft rot pathogen Pectobacterium carotovorum causes a DspE/F-dependent plant cell death on Nicotiana benthamiana within 24 h postinoculation (hpi) followed by leaf maceration within 48 hpi. P. carotovorum strains with mutations in type III secretion system (T3SS) regulatory and structural genes, including the dspE/F operon, did not cause hypersensitive response (HR)-like cell death and or leaf maceration. A strain with a mutation in the type II secretion system caused HR-like plant cell death but no maceration. P. carotovorum was unable to impede callose deposition in N. benthamiana leaves, suggesting that P. carotovorum does not suppress this basal immunity function. Within 24 hpi, there was callose deposition along leaf veins and examination showed that the pathogen cells were localized along the veins. To further examine HR-like plant cell death induced by P. carotovorum, gene expression profiles in N. benthamiana leaves inoculated with wild-type and mutant P. carotovorum and Pseudomonas syringae strains were compared. The N. benthamiana gene expression profile of leaves infiltrated with Pectobacterium carotovorum was similar to leaves infiltrated with a Pseudomonas syringae T3SS mutant. These data support a model where Pectobacterium carotovorum uses the T3SS to induce plant cell death in order to promote leaf maceration rather than to suppress plant immunity.

The Red clover necrotic mosaic virus capsid as a multifunctional cell targeting plant viral nanoparticle.

Multifunctional nanoparticles hold promise as the next generation of therapeutic delivery and imaging agents. Nanoparticles comprising many types of materials are being tested for this purpose, including plant viral capsids. It has been found that Red clover necrotic mosaic virus (RCNMV) can be loaded with significant amounts of therapeutic molecules with molecular weights of 600 or even greater. Formulation of RCNMV into a plant viral nanoparticle (PVN) involves the loading of cargo and attachment of peptides. In this study, we show that targeting peptides (less than 16 amino acids) can be conjugated to the capsid using the heterobifunctional chemical linker sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The uptake of both native RCNMV capsids and peptide-conjugated RCNMV was tested in the HeLa cell line for peptides with and without fluorescent labels. Uptake of RCNMV conjugate with a CD46 targeting peptide was monitored by flow cytometry. When formulated PVNs loaded with doxorubicin and armed with a targeting peptide were delivered to HeLa cells, a cytotoxic effect was observed. The ability to modify RCNMV for specific cell targeting and cargo delivery offers a method for the intracellular delivery of reagents for research assays as well as diagnostic and therapeutic applications.

Targeting cancer with 'smart bombs': equipping plant virus nanoparticles for a 'seek and destroy' mission.

This article discusses plant virus nanoparticles as a weapon in the war on cancer. The successes and failures of numerous nanoparticle strategies are discussed as a background to consideration of the plant virus nanoparticle approach. To have therapeutic benefit, the advantages of the targeted nanoparticle must outweigh the problems of colloidal stability, uptake by the reticuloendothelial system as well as the requirement for clearance from the body. Biodegradable nanoparticles are considered to have the most promise to address these complex phenomena. After justifying the choice of biodegradable particles, the article focuses on comparison of micelles, liposomes, polymers and modified plant viruses. The structural uniformity, cargo capacity, responsive behavior and ease of manufacturing of plant virus nanoparticles are unique properties that suggest they have a wider role to play in targeted therapy. The loading of chemotherapeutic cargo is discussed, with specific reference to the advantage of reversible transitions of the capsid of Red clover necrotic mosaic virus. These features will be contrasted and compared with other biodegradable 'smart bombs' that target cancer cells.

Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.

Nicotiana benthamiana is the most widely used experimental host in plant virology, due mainly to the large number of diverse plant viruses that can successfully infect it. Additionally, N. benthamiana is susceptible to a wide variety of other plant-pathogenic agents (such as bacteria, oomycetes, fungi, and so on), making this species a cornerstone of host-pathogen research, particularly in the context of innate immunity and defense signaling. Moreover, because it can be genetically transformed and regenerated with good efficiency and is amenable to facile methods for virus-induced gene silencing or transient protein expression, N. benthamiana is rapidly gaining popularity in plant biology, particularly in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. Paradoxically, despite being an indispensable research model, little is known about the origins, genetic variation, or ecology of the N. benthamiana accessions currently used by the research community. In addition to addressing these latter topics, the purpose of this review is to provide information regarding sources for tools and reagents that can be used to support research in N. benthamiana. Finally, we propose that N. benthamiana is well situated to become a premier plant cell biology model, particularly for the virology community, who as a group were the first to recognize the potential of this unique Australian native.

A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement.

The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed.

The genomic RNA packaging scheme of Red clover necrotic mosaic virus.

Red clover necrotic mosaic virus (RCNMV) is a small icosahedral plant virus with a bipartite RNA genome. While the RCNMV genome consists of two RNAs, it has not been definitively established whether these RNAs are co-packaged into a single virion or packaged individually into separate virions. Biochemical evidence exists to support both hypotheses. To determine the genomic RNA complement within RCNMV, virions were subjected to heat treatments and UV crosslinking. A stable RNA-1:RNA-2 heterodimer was formed with both treatments establishing that RCNMV genomic RNAs are co-packaged into a single virion. Furthermore, RNA-2 homodimer and homotrimers were also observed indicating that some virions contain multiple copies of RNA-2 exclusively. These results indicate that RCNMV virions consist of two distinct populations: (i) virions containing both genomic RNAs; and (ii) virions with multiple copies of RNA-2. This type of hybrid packaging arrangement was unexpected and appears to be unique among the multipartite RNA viruses.

Cell wall localization of Red clover necrotic mosaic virus movement protein is required for cell-to-cell movement.

The Red clover necrotic mosaic virus movement protein (MP) is essential for cell-to-cell movement. Eight previously characterized alanine-scanning mutants of the MP were fused to the green fluorescent protein (GFP) and expressed from viral infectious transcripts. Inoculated plants were assayed for movement and intracellular accumulation of MP by confocal laser-scanning microscopy. A strict correlation was observed between the targeting to the cell wall (presumably the plasmodesmata) and cell-to-cell movement. Complementation of dysfunctional MP mutants with either wild-type MP or other null mutants in some cases rescued intracellular targeting and movement. The data suggest the presence of distinct domains in the MP for virus movement (near residues 27-31), complementarity (near residues 122 and 128), and intracellular localization (near residue 161). These data support a model of MP interacting cooperatively with itself to bind viral RNA, localize to and modify plasmodesmata and effect virus movement.

Red clover necrotic mosaic virus replication proteins accumulate at the endoplasmic reticulum.

Red clover necrotic mosaic virus (RCNMV) encodes N-terminally overlapping proteins of 27 and 88 kDa (p27 and p88) known to be required for replication. Green fluorescent protein (GFP) fusions were used to visualize the location of p27 and p88 within Nicotiana benthamiana cells. GFP:p27 fusions localized to the endoplasmic reticulum (ER), co-localized with ER-targeted yellow fluorescent protein and caused membrane restructuring and proliferation. Cellular fractionation of virus-inoculated N. benthamiana leaves confirmed the association of p27 with ER membranes. GFP:p88 fusions also localized to the ER and co-localized with GFP:p27. Both fusion proteins co-localize to the cortical and cytoplasmic ER and were associated with invaginations of the nuclear envelope. Independent accumulation in, and perturbation of, the ER suggests that p27 and p88 function together in the replication complex. This is the first report of a member of the Tombusviridae replicating in association with the ER.

The Potato virus Y M(S)N(R) NIb-replicase is the elicitor of a veinal necrosis-hypersensitive response in root knot nematode resistant tobacco.

Summary A root knot nematode resistance gene in Nicotiana tabacum, Rk, providing resistance to the nematode parasite Meloidogyne incognita is tightly linked to, or is a pleiotropic gene with a veinal necrosis systemic hypersensitive response to infection by Potato virus Y (PVY) M(s)N(r). The single PVY M(s)N(r) open reading frame was sequenced and found to have 89% protein identity to PVY N. Individual PVY M(s)N(r) polypeptides were deduced and the corresponding cDNA were cloned into a Potato virus X (PVX) based expression vector and used as templates for in vitro transcriptions. Infected plant sap, from N. benthamiana inoculated with infectious RNA, was used to inoculate both root knot nematode (RKN) resistant and susceptible tobacco lines. Lines were then evaluated for the induction of the hypersensitive response. The PVY M(s)N(r) NIb-replicase protein was found to induce a hypersensitive response 10 days post inoculation in nematode resistant tobacco. None of the other PVX/PVY M(s)N(r) constructs induced a hypersensitive response. The NIb-replicase of PVY N, which shares 93% identity to PVY M(s)N(r), did not induce a hypersensitive response when expressed from the PVX vector. This confirmed that the PVY M(s)N(r) NIb-replicase is the elicitor of PVY M(s)N(r) veinal necrosis on RKN plants and thus the first report of a Potyvirus replicase functioning as an avirulence factor.