Viral nanoparticles for in vivo tumor imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. (1) and (2) for further information on VNPs). From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies. In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Silencing genes encoding omega-3 fatty acid desaturase alters seed size and accumulation of Bean pod mottle virus in soybean.

Omega-3 fatty acid desaturase (FAD3)-catalyzed conversion of linoleic acid to linolenic acid (18:3) is an important step for the biosynthesis of fatty acids as well as the phytohormone jasmonic acid (JA) in plants. We report that silencing three microsomal isoforms of GmFAD3 enhanced the accumulation of Bean pod mottle virus (BPMV) in soybean. The GmFAD3-silenced plants also accumulated higher levels of JA, even though they contained slightly reduced levels of 18:3. Consequently, the GmFAD3-silenced plants expressed JA-responsive pathogenesis-related genes constitutively and exhibited enhanced susceptibility to virulent Pseudomonas syringae. Increased accumulation of BPMV in GmFAD3-silenced plants was likely associated with their JA levels, because exogenous JA application also increased BPMV accumulation. The JA-derived increase in BPMV levels was likely not due to repression of salicylic acid (SA)-derived signaling because the GmFAD3-silenced plants were enhanced in SA-dependent defenses. Furthermore, neither exogenous SA application nor silencing the SA-synthesizing phenylalanine ammonia lyase gene altered BPMV levels in soybean. In addition to the altered defense responses, the GmFAD3-silenced plants also produced significantly larger and heavier seed. Our results indicate that loss of GmFAD3 enhances JA accumulation and, thereby, susceptibility to BPMV in soybean.

Occurrence of Potato virus X on hybrid dock in Czech Republic.

Hybrid dock of Uteush (Rumex patientia L. x Rumex tianschanicus A. Los., the family Polygonaceae) is a perspective high productive crop and in the last decade its farming area has continuously grown in Czech Republic. However, the introduction of this non-native perennial crop into a present plant production creates a new potential reservoir for some plant viruses. Also, the hybrid dock could become a host of currently uncommon or insignificant viruses. We screened two dock-farming localities situated in south-west and north-east part of the Czech Republic for the presence of potyviruses, potexviruses, and carlaviruses. In the south-west part of the country, we detected a high incidence of Potato virus X (PVX, the genus Potexvirus). In contrast, in the north-east part of the country we did not detect any dock plants infected with PVX. Next, two other viruses, Turnip yellow mosaic virus (TYMV) and Radish mosaic virus (RaMV) were mechanically inoculated and tested for their survival capacity and multiplication in the hybrid dock. Both viruses were detected 9 months after inoculation in the infected plants.

Inactivation and purification of cowpea mosaic virus-like particles displaying peptide antigens from Bacillus anthracis.

Chimeric cowpea mosaic virus (CPMV) particles displaying foreign peptide antigens on the particle surface are suitable for development of peptide-based vaccines. However, commonly used PEG precipitation-based purification methods are not sufficient for production of high quality vaccine candidates because they do not allow for separation of chimeric particles from cleaved contaminating species. Moreover, the purified particles remain infectious to plants. To advance the CPMV technology further, it is necessary to develop efficient and scalable purification strategies and preferably eliminate the infectivity of chimeric viruses. CPMV was engineered to display a 25 amino acid peptide derived from the Bacillus anthracis protective antigen on the surface loop of the large coat protein subunit. The engineered virus was propagated in cowpea plants and assembled into chimeric virus particles displaying 60 copies of the peptide on the surface. An effective inactivation method was developed to produce non-infectious chimeric CPMV virus-like particles (VLPs). Uncleaved VLPs were separated from the contaminating cleaved forms by anion exchange chromatography. The yield of purified chimeric VLPs was 0.3 g kg(-1) of leaf tissue. The results demonstrate the ability to generate multi-gram quantities of non-infectious, chimeric CPMV VLPs in plants for use in the development of peptide-based vaccines.

Effect of cowpea seeds contamination rate by the Cowpea aphid borne mosaic virus on epidemics development.

Cowpea aphid borne mosaic virus (CABMV) diseased seeds provide at seedling, virus infected plants which are the only source of primary inoculum. Secondary infections are bequeathed by aphids. The objective of this research is to study the development of the secondary infection in field. Therefore, eight cowpea varieties with different seed contamination rate (0, 0.05, 0.25, 0.5, 1, 5%) were used over consecutive four years. The infected plants were recorded every week from the tenth day after sowing and over seven weeks. In the same way, aphids' population were evaluated in plots 30 days after sowing. There was no difference for the incidence rate between the average of plots sown with virus free-seeds and those sown with infected seeds with a rate of 0, 5%. In any case, the disease progressed lowly leading to incidences less than 50% at the post-flowering period in spite of a relatively high initial contamination rate of seed. For this group of varieties, the low progression of the disease indicated a high level of resistance to the infection. The high levels of infection especially observed with the varieties with high level of virus transmission to seed, translated the need to reduce aphids' population density notably by the use of insecticides during cowpea growing cycle. The high number of aphids and inoculum availability in the neighbouring plots were undoubtedly at the source of this result. This situation laid out the problematic of the use of seeds then little or not contaminated by the virus.

Disruption of leaves and initial extraction of wildtype CPMV virus as a basis for producing vaccines from plants.

Wildtype cowpea mosaic virus (CPMV) was extracted from fresh and frozen plant material by methods suitable for large-scale application. Deep freezing, crushing, and thawing in water or buffers gave 0.6+/-0.2 mg g(-1) of virus after up to 24 h. Release from sliced fresh leaves was lower at 0.14+/-0.03 mg g(-1). Homogenisation of frozen leaves for 1 min increased yield to a maximum, on average of 3.5 mg g(-1) but varying between batches from 2.2 to 4.8 mg g(-1) virus Long term storage at -80 degrees C increased subsequent yield by 2 mg g(-1) per year on average; the maximum was 10.4+/-1.9 mg g(-1) (665 days storage). Within a batch, similar yields were obtained between individual fresh plants, and from frozen versus fresh leaves. After homogenisation for 1 min, 90% of debris particles were smaller than 12 microm, half under 5 microm and 10% less than 1 microm. Homogenate (4% dry weight) was rheologically complex, exhibiting shear thinning with hysteresis at low shear rates which bears on subsequent processing. At shear rates above 200 s(-1), its apparent viscosity was 0.02 N s m(-2).

Mutational analysis of the cowpea mosaic virus movement protein.

Cowpea mosaic virus moves from cell-to-cell in a virion form through tubular structures that are assembled in modified plasmodesmata. Similar tubular structures are formed on the surface of protoplasts inoculated with cowpea mosaic virus. The RNA 2-encoded movement protein (MP) is responsible for the induction and formation of these structures. To define functional domains of the MP, an alanine-substitution mutagenesis was performed on eight positions in the MP, including two conserved sequence motifs, the LPL and D motifs. Results show that these two conserved motifs as well as the central region of the MP are essential for cell-to-cell movement. Several viruses carrying mutations in the N- or C-terminal parts of their MP retained infectivity on cowpea plants. Coexpression studies revealed that mutant MPs did not interfere with the activity of wild-type MP and could not mutually complement their defects.

Cold denaturation of an icosahedral virus. The role of entropy in virus assembly.

Assembly of icosahedral viruses is not completely understood at the molecular level. The main puzzle is to answer how chemically identical protein subunits take up unique positionally dependent conformations during the process of assembly. The stability of the ribonucleoprotein particles of cowpea mosaic virus (CPMV) to pressures and subzero temperatures has been studied. At room temperature, reversible pressure denaturation of CPMV is obtained only in the presence of 5.0 M urea. On the other hand, when the temperature is decreased to -15 degrees C, the ribonucleoprotein components denature, at 2.5 kbar, in the presence of 1.0 M urea. At temperatures close to -20 degrees C, denaturation is obtained even in the absence of urea. Whereas the denaturation promoted by pressure and urea at room temperature is reversible, virus particles denatured when the temperature is decreased under pressure cannot reassemble. Bis-ANS binding data suggest that this irreversibility may be related to protein release from RNA, which probably does not occur under denaturating conditions at room temperature. The contributions of enthalpy (delta H*) and entropy (delta S*) for the free energy of association of CPMV are calculated from the cold denaturation curves under pressure. The entropy change is positive and large, making the assembly of ribonucleoprotein components an entropy-driven process, suggesting that the burial of nonpolar side chains during the process of assembly is the structural foundation for CPMV assembly.

Replication and translation of cowpea mosaic virus RNAs are tightly linked.

The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RNA is able to replicate independently from M-RNA in cowpea protoplasts. Replication of mutant B-transcripts could not be supported by co-inoculated wild-type B-RNA, indicating that B-RNA cannot be efficiently replicated in trans. Hence replication of a B-RNA molecule is tightly linked to its translation and/or at least one of the replicative proteins functions in cis only. Remarkably also for efficient replication of M-RNA one of its translation products was found to be required in cis. This 58K protein possibly helps in directing the B-RNA-encoded replication complex to the M-RNA. In order to identify the viral polymerase the CPMV B-RNA-specific proteins have been produced individually in cowpea protoplasts using CaMV 35S promoter based expression vectors. Only protoplasts transfected with a vector containing the 200K coding sequence were able to support replication of co-transfected M-RNA. Despite this, CPMV-specific RNA polymerase activity could not be detected in extracts of these protoplasts using a poly(A)/oligo(U) assay. These results indicate that, in contrast to the poliovirus polymerase, the CPMV polymerase is not able to accept oligo(U) as a primer and in addition support the concept that translation and replication are linked.