The generation of empty Turnip yellow mosaic virus capsids through depletion of virion-associated divalent cations.

Turnip yellow mosaic virus (TYMV) is a well-studied icosahedral plant virus that has attractive properties for nanoscience applications. Stable empty particles devoid of viral genomic RNA have historically been generated from virions by: 1. high pressure; 2. extreme alkaline pH; and 3. freeze-thaw using liquid nitrogen. Herein we report a fourth and more convenient avenue for empty particle formation through EDTA treatment, implicating chelation of virion-associated cations. We present findings that confirm TYMV virions purified in an EDTA-based buffer are converted to 94 % empty on average during purification. Additional experimentation revealed TYMV virions purified through CsCl vs. sucrose gradients are more readily converted to empty particles after freeze thaw. These studies are novel as they show a purification method through EDTA-treatment that can generate stable empty particles devoid of viral genome. The convenience of this method should prove suitable for scientists seeking to use TYMV capsids in nanoscience-inspired applications. Importantly, these findings provide insight into historical discrepancies in creating empty particles after freeze-thaw, as the method in which TYMV virions are purified influences the downstream virion-to-empty conversion process.

Cisplatin Prodrug-Loaded Nanoparticles Based on Physalis Mottle Virus for Cancer Therapy.

Nanoparticle-based prodrugs offer an effective strategy to improve the safety and delivery of small-molecule therapeutics while reducing the risk of drug resistance. Here, we conjugated a maleimide-functionalized cisplatin prodrug containing Pt(IV) to the internal and/or external surface of virus-like particles (VLPs) derived from Physalis mottle virus (PhMV) to develop a pH-sensitive drug delivery system. The internally loaded and PEGylated VLPs (Pt-PhMVCy5.5-PEG) were taken up efficiently by cancer cells where they released platinum, presumably as a reduced, DNA-reactive Pt(II) complex, rapidly under acidic conditions in vitro (>80% in 30 h). The efficacy of the VLP-based drug delivery system was demonstrated against a panel of cancer cell lines, including cell lines resistant to platinum therapy. Furthermore, Pt-PhMVCy5.5-PEG successfully inhibited the growth of xenograft MDA-MB-231 breast tumors in vivo and significantly prolonged the survival of mice compared to free cisplatin and cisplatin-maleimide. Pt-PhMVCy5.5-PEG therefore appears promising as a prodrug to overcome the limitations of conventional platinum-based drugs for cancer therapy.

Physalis Mottle Virus-like Nanoparticles for Targeted Cancer Imaging.

One of the greatest challenges in nanomedicine is the low efficiency with which nanoparticles are delivered to lesions such as tumors in vivo. Here, we show that Physalis mottle virus (PhMV)-like nanoparticles can be developed as bimodal contrast agents to achieve long circulation, specific targeting capability, and efficient delivery to tumors in vivo. The self-assembling coat protein nanostructure offers various opportunities to modify the internal and external surfaces separately. After loading the internal cavity of the particles with the fluorescent dye Cy5.5 and paramagnetic Gd(III) complexes, we modified the outer surface by PEGylation and conjugation with targeting peptides. Using this combined approach, we were able to monitor a human prostate tumor model for up to 10 days by near-infrared fluorescence and magnetic resonance imaging, with up to 6% of the injection dose remaining. Our results show that PhMV-like nanoparticles provide a promising and innovative platform for the development of next-generation diagnostic and therapeutic agents.

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.

Physalis Mottle Virus-Like Particles as Nanocarriers for Imaging Reagents and Drugs.

Platform technologies based on plant virus nanoparticles (VNPs) and virus-like particles (VLPs) are attracting the attention of researchers and clinicians because the particles are biocompatible, biodegradable, noninfectious in mammals, and can readily be chemically and genetically engineered to carry imaging agents and drugs. When the Physalis mottle virus (PhMV) coat protein is expressed in Escherichia coli, the resulting VLPs are nearly identical to the viruses formed in vivo. Here, we isolated PhMV-derived VLPs from ClearColi cells and carried out external and internal surface modification with fluorophores using reactive lysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries, respectively. The uptake of dye-labeled particles was tested in a range of cancer cells and monitored by confocal microscopy and flow cytometry. VLPs labeled internally on cysteine residues were taken up with high efficiency by several cancer cell lines and were colocalized with the endolysosomal marker LAMP-1 within 6 h, whereas VLPs labeled externally on lysine residues were taken up with lower efficiency, probably reflecting differences in surface charge and the propensity to bind to the cell surface. The infusion of dye and drug molecules into the cavity of the VLPs revealed that the photosensitizer (PS), Zn-EpPor, and the drugs crystal violet, mitoxantrone (MTX), and doxorubicin (DOX) associated stably with the carrier via noncovalent interactions. We confirmed the cytotoxicity of the PS-PhMV and DOX-PhMV particles against prostate cancer, ovarian and breast cancer cell lines, respectively. Our results show that PhMV-derived VLPs provide a new platform technology for the delivery of imaging agents and drugs, with preferential uptake into cancer cells. These particles could therefore be developed as multifunctional tools for cancer diagnosis and therapy.

A mobile loop near the active site acts as a switch between the dual activities of a viral protease/deubiquitinase.

The positive-strand RNA virus Turnip yellow mosaic virus (TYMV) encodes an ovarian tumor (OTU)-like protease/deubiquitinase (PRO/DUB) protein domain involved both in proteolytic processing of the viral polyprotein through its PRO activity, and in removal of ubiquitin chains from ubiquitylated substrates through its DUB activity. Here, the crystal structures of TYMV PRO/DUB mutants and molecular dynamics simulations reveal that an idiosyncratic mobile loop participates in reversibly constricting its unusual catalytic site by adopting "open", "intermediate" or "closed" conformations. The two cis-prolines of the loop form a rigid flap that in the most closed conformation zips up against the other side of the catalytic cleft. The intermediate and closed conformations also correlate with a reordering of the TYMV PRO/DUB catalytic dyad, that then assumes a classical, yet still unusually mobile, OTU DUB alignment. Further structure-based mutants designed to interfere with the loop's mobility were assessed for enzymatic activity in vitro and in vivo, and were shown to display reduced DUB activity while retaining PRO activity. This indicates that control of the switching between the dual PRO/DUB activities resides prominently within this loop next to the active site. Introduction of mutations into the viral genome revealed that the DUB activity contributes to the extent of viral RNA accumulation both in single cells and in whole plants. In addition, the conformation of the mobile flap was also found to influence symptoms severity in planta. Such mutants now provide powerful tools with which to study the specific roles of reversible ubiquitylation in viral infection.

Crystallization of mutants of Turnip yellow mosaic virus protease/ubiquitin hydrolase designed to prevent protease self-recognition.

Processing of the polyprotein of Turnip yellow mosaic virus is mediated by the protease PRO. PRO cleaves at two places, one of which is at the C-terminus of the PRO domain of another polyprotein molecule. In addition to this processing activity, PRO possesses an ubiquitin hydrolase (DUB) activity. The crystal structure of PRO has previously been reported in its polyprotein-processing mode with the C-terminus of one PRO inserted into the catalytic site of the next PRO, generating PRO polymers in the crystal packing of the trigonal space group. Here, two mutants designed to disrupt specific PRO-PRO interactions were generated, produced and purified. Crystalline plates were obtained by seeding and cross-seeding from initial `sea urchin'-like microcrystals of one mutant. The plates diffracted to beyond 2 Šresolution at a synchrotron source and complete data sets were collected for the two mutants. Data processing and analysis indicated that both mutant crystals belonged to the same monoclinic space group, with two molecules of PRO in the asymmetric unit.

An RNA tertiary switch by modifying how helices are tethered.

A viral tRNA-like structure has evolved a unique strategy to undergo a tertiary structure conformational switch that may help regulate viral regulation.

A synthetic nanomaterial for virus recognition produced by surface imprinting.

Major stumbling blocks in the production of fully synthetic materials designed to feature virus recognition properties are that the target is large and its self-assembled architecture is fragile. Here we describe a synthetic strategy to produce organic/inorganic nanoparticulate hybrids that recognize non-enveloped icosahedral viruses in water at concentrations down to the picomolar range. We demonstrate that these systems bind a virus that, in turn, acts as a template during the nanomaterial synthesis. These virus imprinted particles then display remarkable selectivity and affinity. The reported method, which is based on surface imprinting using silica nanoparticles that act as a carrier material and organosilanes serving as biomimetic building blocks, goes beyond simple shape imprinting. We demonstrate the formation of a chemical imprint, comparable to the formation of biosilica, due to the template effect of the virion surface on the synthesis of the recognition material.

Crystallization, structural diversity and anisotropy effects in 2D arrays of icosahedral viruses.

We investigate two-dimensional (2D) assembly of the icosahedral turnip yellow mosaic virus (TYMV) under cationic lipid monolayers at the aqueous solution­vapor interface. The 2D crystallization of TYMV has been achieved by enhancing electrostatically induced interfacial adsorption, an approach recently demonstrated for another virus. In situ X-ray scattering reveals two close-packed 2D crystalline phases of TYMV that are distinct from the previously reported hexagonal and centered square (√2 × âˆš2) arrays of TYMV. One of the newly observed phases arises from either a dimeric double-square (2 × 1) or tetrameric square (2 × 2) unit cell. The other is a rhombic crystal with a lattice angle of 80°. The two observed crystal phases are substantially less dense (by over 10%) than a 2D lattice of TYMV could be according to its known size and shape, indicating that local anisotropic interparticle interactions play a key role in stabilizing these crystals. TYMV's anisotropy attributes and numerical analysis of 2D arrays of virus-shaped particles are used to derive a model for the rhombic crystal in which the particle orientation is consistent with the electrostatic lipid­TYMV attraction and the interparticle contacts exhibit steric complementarity. The interplay between particle anisotropy and packing is contrasted between the rhombic crystal model and the square (√2 × âˆš2) crystal. This study highlights how the high symmetry and subtle asphericity of icosahedral particles enrich the variety and complexity of ordered 2D structures that can be generated through self-assembly.

Polyvalent display of RGD motifs on turnip yellow mosaic virus for enhanced stem cell adhesion and spreading.

Turnip yellow mosaic virus (TYMV) is a stable 28 nm icosahedral plant virus that can be isolated in gram quantities. In order to study the polyvalent effect of Arg-Gly-Asp (RGD) clustering on the response of bone marrow stem cells (BMSCs), an RGD motif was genetically displayed on the coat protein of the TYMV capsid. Composite films composed of either wild-type TYMV or TYMV-RGD44, in combination with poly(allylamine hydrochloride) (PAH), were fabricated by a layer-by-layer adsorption of virus and PAH. The deposition process was studied by quartz crystal microbalance, UV-visible spectroscopy and atomic force microscopy. BMSC adhesion assays showed enhanced cell adhesion and spreading on TYMV-RGD44 coated substrates compared to native TYMV. These results demonstrate the potential of TYMV as a viable scaffold for bioactive peptide display and cell culturing studies.

Chemoselective modification of turnip yellow mosaic virus by Cu(I) catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction and its application in cell binding.

Turnip yellow mosaic virus (TYMV) is an icosahedral plant virus with a diameter of 28-30 nm that can be isolated in gram quantities from turnip or Chinese cabbage inexpensively. In this study, TYMV combined with spatially addressable surface chemistries was selected as a prototype bionanoparticle for modulating patterns of cell adhesion, morphology, and proliferation. We exploited the chemical reactivity of TYMV using the mild conditions of Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction, the best example of "click" chemistry. Oligo-ethylene glycol (OEG) short chain, coumarintriazole, and RGD-containing peptide were grafted on the surface of TYMV via carbodiimide activation and CuAAC reaction. The bioconjugation to intact viral particles was confirmed by MS, TEM, FPLC, and SDS-PAGE with fluorescence visualization analysis. Therefore, this method is a generally useful means of incorporating various types of functionalities onto the TYMV surface. Further studies were done to learn the behavior of NIH-3T3 fibroblast cells on the modified or unmodified TYMV surfaces. OEG-modified TYMV surfaces retarded cell attachment and growth, while cell adhesion, spreading, and proliferation were dramatically enhanced on RGD-modified TYMV surfaces. Compared with RGD immobilized 3-aminopropyltriethoxysilane-coated glass surface, the cells are more ready to spread fully and proliferate on TYMV-RGD coated surface, which thus provides a more cell-friendly environment with nanometer-scale surface features. This illustrates the potential application of plant virus based materials in tissue engineering, drug delivery, and biosensing.

Interfacial assembly of turnip yellow mosaic virus nanoparticles.

An extensive study of the factors that affect the interfacial assembly of bionanoparticles at the oil/water (O/W) interface is reported. Bionanoparticles, such as viruses, have distinctive structural properties due to the unique arrangement of their protein structures. The assembly process of such bionanoparticles at interfaces is governed by factors including the ionic strength and pH of the aqueous layer, concentration of the particles, and nature of the oil phase. This study highlights the impact of these factors on the interfacial assembly of bionanoparticles at the O/W interface using native turnip yellow mosaic virus (TYMV) as the prototype. Robust monolayer assemblies of TYMV were produced by self-assembly at the O/W interface using emulsions and planar interfaces. TYMV maintained its structure and integrity under different assembly conditions. For the emulsion droplets, they were fully covered with TYMV as evidenced by transmission electron microscopy (TEM) and scanning force microscopy (SFM). Tensiometry and small-angle neutron scattering (SANS) further supported this finding. Although the emulsions offered a complete coverage by TYMV particles, they lacked long-range ordering due to rapid exchange at the interface. By altering the assembly process, highly ordered, hexagonal arrays of TYMV were obtained at planar O/W interfaces. The pH, ionic strength, and viscosity of the solution played a crucial role in enhancing the lateral ordering of TYMV assembled at the planar O/W interface. This interfacial ordering of TYMV particles was further stabilized by introduction of a positively charged dehydroabietyl amine (DHAA) in the organic phase which held the assembly together by electrostatic interactions. The long-range array formation was observed using TEM and SFM. The results presented here illustrate that the interfacial assembly at the O/W interface is a versatile approach to achieve highly stable self-assembled structures.

Turnip yellow mosaic virus as a chemoaddressable bionanoparticle.

Viruses and virus-like particles (VLPs) have been demonstrated to be robust scaffolds for the construction of nanomaterials. In order to develop new nanoprobes for time-resolved fluoroimmuno assays as well as to investigate the two-dimensional self-assembly of viruses and VLPs, the icosahedral turnip yellow mosaic virus (TYMV) was investigated as a potential building block in our study. TYMV is an icosahedral plant virus with an average diameter of 28 nm that can be isolated inexpensively in gram quantities from turnips or Chinese cabbage. There are 180 coat protein subunits per TYMV capsid. The conventional N-hydroxysuccinimide-mediated amidation reaction was employed for the chemical modification of the viral capsid. Tryptic digestion with sequential MALDI-TOF MS analysis identified that the amino groups of K32 of the flexible N-terminus made the major contribution for the reactivity of TYMV toward N-hydroxysuccinimide ester (NHS) reagents. The reactivity was also monitored with UV-vis absorbance and fluorescence, which revealed that approximately 60 lysines per particle could be addressed. We hypothesized that the flexible A chain contains the reactive lysine because the crystal structure of TYMV has shown that chain A is much more flexible compared to B and C, especially at the N-terminal region where the Lys-32 located. In addition, about 90 to 120 carboxyl groups, located in the most exposed sequence, could be modified with amines catalyzed with 1-(3-dimethylaminopropyl-3-ethylcarbodiimide) hydrochloride (EDC) and sulfo-NHS. TYMV was stable to a wide range of reaction conditions and maintained its integrity after the chemical conjugations. Therefore, it can potentially be employed as a reactive scaffold for the display of a variety of materials for applications in many areas of nanoscience.

Patterns of recombination in turnip mosaic virus genomic sequences indicate hotspots of recombination.

Potyviruses have variable single-stranded RNA genomes and many show clear evidence of recombination. This report studied the distribution of recombination sites in the genomes of 92 isolates of the potyvirus Turnip mosaic virus (TuMV); 42 came from the international gene sequence databases and an additional 50 complete genomic sequences were generated from field samples collected in Europe and Asia. The sequences were examined for evidence of recombination using seven different sequence comparison methods and the exact position of each site was confirmed by sequence composition analysis. Recombination sites were found throughout the genomes, except in the small 6K1 protein gene, and only 24 of the genomes (26%) showed no evidence of recombination. Statistically significant clusters of recombination sites were found in the P1 gene and in the CI/6K2/VPg gene region. Most recombination sites were bordered by an upstream (5') region of GC-rich and downstream (3') region of AU-rich sequence of a similar length. Correlations between the presence and type of recombination site and provenance, host type and phylogenetic relationships are discussed, as is the role of recombination in TuMV evolution.

The role of inter-subunit ionic interactions in the assembly of Physalis mottle tymovirus.

Physalis mottle tymovirus (PhMV) is a small spherical plant virus with its RNA genome encapsidated in a protein shell made of 180 identical coat protein (CP) subunits. The amino acid residues involved in two interfacial salt bridges, Asp-83/Arg-159 and Arg-68/Asp-150 and Lys-153, were targeted for mutagenesis with a view to delineate the role of interfacial ionic interactions in the subunit folding and assembly of the virus. R159A and D83A-R159A recombinant CP (rCP) mutants formed stable T = 3 capsids, indicating that the D83-R159 interfacial salt bridge is dispensable for the folding and assembly of PhMV. However, D150A and R68Q-D150A mutant rCPs were present in the insoluble fraction, suggesting that the R68-D150 interfacial salt bridge is crucial for subunit folding and assembly. Similarly, K153Q, D83A-K153Q, and H69A-K153Q mutant rCPs were present in the insoluble fraction. Interestingly, the R68Q-D150A, D83A-K153Q, and H69A-K153Q double mutant rCPs could be refolded into partially folded soluble heterogeneous aggregates of 14-16 S. The results further confirm our earlier observation that subunit folding and assembly are concerted events in PhMV.

Atomic force microscopy investigation of Turnip Yellow Mosaic Virus capsid disruption and RNA extrusion.

Turnip Yellow Mosaic Virus (TYMV) was subjected to a variety of procedures which disrupted the protein capsids and produced exposure of the ssRNA genome. The results of the treatments were visualized by atomic force microscopy (AFM). Both in situ and ex situ freeze-thawing produced RNA emission, though at low efficiency. The RNA lost from such particles was evident, in some cases in the process of exiting the virions. More severe disruption of TYMV and extrusion of intact RNA onto the substrate were produced by drying the virus and rehydrating with neutral buffer. Similar products were also obtained by heating TYMV to 70 -75 degrees C and by exposure to alkaline pH. Experiments showed the nucleic acid to have an elaborate secondary structure distributed linearly along its length.

Variation in the coat protein sequence of British isolates of Turnip yellow mosaic virus and comparison with previously published isolates. Brief report.

Isolates of Turnip yellow mosaic virus (TYMV) were collected from wild cabbage (Brassica oleracea) on a 400 m stretch of Dorset coastline. The coat protein genes of four isolates showed high homology in nucleotide sequence (0.970-1.000, mean 0.987). Lower levels of homology where found to previously published sequences of Australian isolates [10] (0.725-0.775, mean 0.741). The amino acid composition of the Dorset isolates showed high levels of homology (0.964-1.000, mean 0.986). Numerous amino acid substitutions occurred between the Dorset and Australian isolates (0.705-0.819, mean 0.742). Comparison with other isolates showed large genetic distances between the Dorset isolates and both European and Australian isolates.

Nemesia ring necrosis virus: a new tymovirus with a genomic RNA having a histidylatable tobamovirus-like 3' end.

The complete nucleotide sequence of the genomic RNA of the new virus Nemesia ring necrosis virus (NeRNV), which is widespread in various ornamental plant species belonging to the Scrophulariaceae and Verbenaceae, has been determined. Based on its gene content, the folding properties of its 5'-untranslated region and in vitro translation experiments, NeRNV RNA is a typical tymovirus RNA. Its 3' end, however, differs greatly from those of the valine-specific tymoviral RNAs that have been analysed previously. It can be folded into an upstream pseudoknot domain and a histidine-specific tRNA-like structure, a combination that, so far, has been found only in tobamoviral RNAs. The identity elements found in NeRNV RNA for recognition by yeast histidyl-tRNA synthetase are more similar to those of yeast tRNAHis than the ones found in tobacco mosaic virus RNA. As a result NeRNV RNA can be charged with histidine even more efficiently than tobacco mosaic virus RNA.

The RNA of turnip yellow mosaic virus exhibits icosahedral order.

Difference electron density maps, based on structure factor amplitudes and experimental phases from crystals of wild-type turnip yellow mosaic virus and those of empty capsids prepared by freeze-thawing, show a large portion of the encapsidated RNA to have an icosahedral distribution. Four unique segments of base-paired, double-helical RNA, one to two turns in length, lie between 33-A and 101-A radius and are organized about either 2-fold or 5-fold icosahedral axes. In addition, single-stranded loops of RNA invade the pentameric and hexameric capsomeres where they contact the interior capsid surface. The remaining RNA, not seen in electron density maps, must serve as connecting links between these secondary structural elements and is likely icosahedrally disordered. The distribution of RNA observed crystallographically appears to be in agreement with models based on biochemical data and secondary structural analyses.