Tobacco Mosaic Virus Particles Research Articles

The Presence of Tobacco Mosaic Virus in the Compost Extract of Cigar Tobacco Debris

Tobacco mosaic virus (TMV) is resistance to high temperature and able to survive over 10 years on dried leaves, and plant debris is considered as source of inoculums of TMV in the field. In order to inactivate TMV, TMV-infected cigar tobacco debris was composted at starting temperature of 50 °C for two to three days; however, TMV was still infective in the extract compost. If a half leaf cigar tobacco ‘H877’ was inoculated with compost extract, the symptoms appeared as a necrotic local lesion (NLL) and did not develop systemic lesions. The dilution end point of TMV in extract compost was 10 −3 . The number of lesion was higher in the glasshouse with average daylight temperature of 32 °C than in the field with average daylight temperature of 29–30 °C. The number NLL was lower and NLL size seemed to be smaller on the first and second inoculated leaves with extract than that of on the first and second inoculated leaves with TMV inoculums. There was a delay of time about 58–106 hours after inoculation of NLL from extract compost inoculums to appear than those of from TMV inoculums. These could be happened because of mineral nutrients of compost and also the temperature of maintaining tobacco plant which inhibited the infections, and of a thermal composting process which destroyed some TMV particles, particularly degraded it's coat protein.

Layer‐By‐Layer Assembly of Viral Nanoparticles and Polyelectrolytes: The Film Architecture is Different for Spheres Versus Rods

The development of tuneable thin film assemblies that contain (bio)nanoparticles is an emerging field in nanobiosciences/nanotechnology. Our research focuses on the utilisation of viral nanoparticles (VNPs) as tools and building blocks for materials science. In previous reports we studied multilayered arrays of chemically modified cowpea mosaic virus (CPMV) particles and linker molecules. To extend these studies and to gain more insights into the architecture of the arrays, we report here on the construction of multilayered assemblies of native plant viral particles and polyelectrolytes. We specifically addressed the question of whether the shape of the VNPs influences the overall structures of the arrays. To study this, we have chosen two particles with similar surface properties but different shapes: CPMV was used as a sphere-like VNP, and tobacco mosaic virus (TMV) served as a rod-shaped VNP. The multilayers were self-assembled on solid supports through electrostatic interactions. Multilayer build-up was followed by quartz crystal microbalance with dissipation monitoring and UV/Vis spectroscopy. Scanning electron microscopy was used to characterize the topologies of the thin films. Our studies show that shape indeed matters. Incorporation of CPMV in alternating arrays of VNPs and polyelectrolytes is demonstrated; in stark contrast, TMV particles were found to be excluded from the arrays, and floated atop the architecture in an ordered structure.

Nano ES GEMMA and PDMA, New Tools for the Analysis of Nanobioparticles—Protein Complexes, Lipoparticles, and Viruses

Differential mobility analysis (DMA) is a technique suited for size analysis as well as preparative collection of airborne nanosized airborne particles. In the recent decade, the analysis of intact viruses, proteins, DNA fragments, polymers, and inorganic nanoparticles was possible when combining this method with a nano-electrospray charge-reduction source for producing aerosols from a sample solution/suspensions. Mass analysis of high molecular weight noncovalent complexes is also possible with this methodology due to the linear correlation of the electrophoretic mobility diameter and the molecular mass. In this work, we present the analysis (size and molecular mass) of high molecular weight multimers (noncovalent functional homocomplex) of Jack bean urease in a mass range from 275 kDa up to 2.5 MDa, with mainly present tri- and hexamers but also higher oligomers of the 91 kDa monomer subunit. In a second experiment, the size analysis of intact very-low-density (approximately 35 nm), low-density ( approximately 22 nm) and high-density lipoparticles (approximately 10 nm), which are heterocomplexes consisting of cholesterol, lipids, and proteins in different ratios, is presented. Results from mobility analysis were in excellent agreement with particle diameters found in literature. The last presented experiment demonstrates size analysis of a rod-like virus and selective sampling of a selected size fraction of electrosprayed, singly-charged tobacco mosaic virus particles. Sampling and subsequent transmission electron microscopic investigations of a specific size fraction (40 nm electrophoretic mobility diameter) revealed the folding of virus particles during the electrospray and charge reduction (electrical stress) as well as solvent evaporation (mechanical stress) process, leading to an observed geometry of 150 (length) x 35 (width) nm (average cylindrical geometry of unsprayed intact virus 300 x 18 nm).

Coagulation of tobacco mosaic virus in alcohol–water–LiCl solutions

The coagulation and colloidal stability of tobacco mosaic virus (TMV) in alcohol–water–LiCl solutions were studied. Without the addition of LiCl salt, the coagulation was promoted by the increase of hydrophobicity of the alcohols that is proportional to their alkyl chain length and concentration. Addition of the LiCl salt reduced the electrostatic repulsion between TMV particles resulting in coagulation in methanol–water and ethanol–water solutions. In water–alcohol–LiCl mixture, the coagulation of TMV was driven by both the hydrophobic interaction of the solution and the screening effect of the salt simultaneously. To understand the particle–particle interaction during the coagulation, the interaction energy was calculated using DLVO theory. Considering the electrostatic repulsive energy, van der Waals attractive energy, and hydrophobic interaction energy, the total energy profiles were obtained. The experiment and model calculation results indicated that the increase of alcohol concentration would increase hydrophobic attraction energy so that the coagulation is promoted. These results provide the fundamental understanding on the coagulation of biomolecular macromolecules.

Use of surface plasmon resonance imaging to study viral RNA:protein interactions

Surface plasmon resonance imaging (SPRi) is an emerging microarray technology that is label-free, rapid and extremely flexible. Here the capabilities of SPRi are demonstrated in results of proof-of-concept experiments detailing a method for studying viral genomic RNA:protein interactions in array format. The principal RNA is the well-characterized origin of assembly (OAS) containing region of Tobacco mosaic virus (TMV) RNA, whereas the principal protein is the primary subunit for TMV virion assembly, the 20S capsid protein aggregate. DNA probes complementary to TMV and non-TMV RNA fragments were covalently attached to a thin gold layer deposited on glass. These DNA probes were used to discreetly capture in vitro transcribed TMV and Red clover necrotic mosaic virus (RCNMV) RNA2 (used as a negative control for the subsequent protein binding). The 4S TMV capsid protein monomers were isolated from TMV particles purified from infected plants of Nicotiana tabacum L. and were induced to form 20S stacked disc aggregates. These 20S stacked disc aggregates were then injected onto the array containing the RNA fragments captured by the DNA probes immobilized on the microarray surface. The discrete and preferential binding of the 20S stacked disc aggregates to the array locations containing the TMV OAS RNA sequence was observed. The results demonstrate that SPRi can be used to monitor binding of large RNA molecules to immobilized DNA capture probes which can then be used to monitor the subsequent binding of complex protein structures to the RNA molecules in a single real-time, label-free microarray experiment. The results further demonstrate that SPRi can distinguish between RNA species that have or do not have an origin of assembly sequence specific for a particular viral capsid protein or protein complex. The broader implications of these results in virology research are found in other systems where the research goals include characterizing the specificity and kinetics of viral or host protein or protein complex interactions with viral nucleic acids.

Copper nanowires within the central channel of tobacco mosaic virus particles

Simple electrochemical deposition techniques can produce highly defined metal nanostructures in templates. Electroless deposition (ELD) can be effectively used for depositing metals on insulators such as biological or plastic surfaces. With biomolecular templates, metallization methods are often restricted to mild reductions, and the deposition of copper at pH values above 12 is usually not applicable. We produced copper nanowires of 3 nm in diameter and up to 150 nm in length by electroless deposition within the 4 nm wide channel of tobacco mosaic virus (TMV) particles. We employed a low pH (∼7.5) copper electroless deposition solution that is compatible with biomolecules. The fabrication process of the nanowires is based on sensitization of tobacco mosaic virus with Pd(II) prior to the electroless deposition. We analyzed the chemical composition of the nanowires by energy-filtering transmission electron microscopy, and used the method also for nickel and cobalt nanowires deposited within the viral channel.

Protection of rabbits against cutaneous papillomavirus infection using recombinant tobacco mosaic virus containing L2 capsid epitopes

Cottontail rabbit papillomavirus (CRPV) and rabbit oral papillomavirus (ROPV) represent distantly related, cutaneous and mucosal tissue tropic papillomaviruses respectively that can infect the same host. These two viruses were used to test the effectiveness of an L2 peptide-based vaccine (aa 94-122) that was delivered on the surface of recombinant tobacco mosaic virus (rTMV) particles. Groups of NZW rabbits received combinations of CRPVL2, ROPVL2 and CRPV + ROPVL2 rTMV vaccines, and were then challenged with infectious CRPV and ROPV. The rabbits developed antibodies that reacted to whole L2 protein and these sera were able to neutralize CRPV pseudovirions at half-maximal titers that were between 50 and 500. Rabbits receiving the CRPV L2 vaccine alone or in combination with ROPV L2 vaccines were completely protected against CRPV infections. Those rabbits vaccinated with the ROPV L2 vaccines showed a weak response in some rabbits against CRPV infection. These studies demonstrate that L2-based vaccines provide strong protection against experimental papillomavirus infection that is most likely based upon the induction of virus-neutralizing antibody. Notably, we observed some limited cross-protection induced by the L2 sequences tested in these vaccines. Finally, the study demonstrated that rTMV were excellent agents for the induction of strong protection in a pre-clinical disease model of papillomavirus infection.

In Planta production of immunogenic poliovirus peptide using tobacco mosaic virus-based vector system

The tobacco mosaic virus (TMV) provides an attractive means of producing foreign peptides in plants. In this study, a TMV-based vector was designed such that a fragment encoding 15 amino acids of the poliovirus peptide (PVP) derived from the viral capsid proteins VP3 and VP1 of poliovirus type 1 Sabin was inserted downstream of the six-base 3' context nucleotide sequence of the TMV coat protein (CP) gene. This design allowed readthrough at the amber stop codon, thereby producing the chimeric TMV particle with both intact CP and CP-fusion protein (CP-PVP) in Nicotiana tabacum cv. Samsun infected with the TMV vector. The TMVCP-PVP virus particle induced antibodies against PVP as well as TMVCP in mice after intraperitoneal immunization. These data illustrate the potential of the readthrough translation system with TMVCP for antigen presentation and vaccine production.

Modified Tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications

Display of peptides or proteins in an ordered, repetitive array, such as on the surface of a virus-like particle, is known to induce an enhanced immune response relative to vaccination with the “free” protein antigen. The coat protein of Tobacco mosaic virus (TMV) can accommodate short peptide insertions into the primary sequence, but the display of larger protein moieties as genetic fusions to the capsid protein has not been possible. We employed a randomized library approach to introduce a reactive lysine at the externally located amino terminus of the coat protein, which facilitated biotinylation of the capsid. To characterize display of heterologous proteins on the virion surface, we bound a model antigen (green fluorescent protein (GFP)–streptavidin (SA), expressed and purified from plants) to the biotinylated TMV particles, creating a GFP-SA decorated virus particle. A GFP-SA tetramer loading of 26% was obtained, corresponding to approximately 2200 GFP moieties displayed per intact virion. We evaluated the immunogenicity of GFP decorated virions in both mice and guinea pigs and found augmented humoral IgG titers in both species, relative to unbound GFP-SA tetramer. Next, we fused an N-terminal fragment of the Canine oral papillomavirus L2 protein to streptavidin. With TMV display, the L2 protein fragment was significantly more immunogenic than uncoupled antigen when tested in mice. By demonstrating the presentation of whole proteins, this study expands the utility of TMV as a vaccine scaffold beyond that which is possible by genetic manipulation.

Characterization of silica-coated tobacco mosaic virus

The deposition of silica on the surface of tobacco mosaic virus (TMV) is achieved at a higher pH (>7) as a means to enhance its usefulness as a template for the synthesis of nanostructures. Electron energy loss spectroscopy definitively shows the presence of a silica shell on the surface of the TMV while small angle X-ray scattering differentiates successfully between silica-coated TMV and silica particles in the presence of uncoated TMV. Importantly, coating reactions done in a 50% w/v methanol/water solution produce smaller silica nanostructures during the condensation of the hydrolysis intermediates, possibly aiding in obtaining uniform coating. Furthermore, TMV-templated silica coatings are found to enhance the stability of the virus particle in methanol at conditions that would ordinarily disrupt the assembled particle. Combined these findings demonstrate that TMV can function as an efficient template for the controlled deposition of silica at neutral pH.

Effect of CuCl 2 concentration on the aggregation and mineralization of Tobacco mosaic virus biotemplate

Aggregation of the biotemplates in mineralization processes is a considerable obstacle in preparing well-dispersed bio-inorganic hybrid materials. In this study, aggregation and mineralization of Tobacco mosaic virus (TMV) biotemplates were investigated as a function of the copper precursor (CuCl 2) concentration. The mean hydrodynamic radius of TMV in an aqueous CuCl 2 solution was determined by dynamic light scattering for the monitoring of the TMV aggregation. At CuCl 2 concentration of 0.5 mM or higher, the mean hydrodynamic radius of TMV increased dramatically indicating aggregation of the TMV particles. Numerical calculations on the long-range interaction energy between parallel model TMV particles agreed with the experimental observations for the TMV aggregation. Mineralization of copper precursors on the TMV biotemplates was achieved only at the CuCl 2 concentrations that induced considerable aggregation of the biotemplate. From the numerical calculations and experimental results, it was concluded that a dense copper cluster deposition cannot be achieved without aggregation of TMV templates.

Using the surface spontaneous depolarization field of ferroelectrics to direct the assembly of virus particles

Tobacco mosaic virus (TMV) particles have been assembled at predetermined locations on the surface of a ferroelectric thin film with the composition PbZr0.3Ti0.7O3. The domain structure, hence the surface depolarization field for the ferroelectric associated with the bending of the ferroelectric band structure, was modified at length scales of around 2μm. A suspension of TMV particles was deposited onto the surface of the ferroelectric over the region where domain modification had been performed. SPM images (intermittent contact) revealed that the virus particles were attracted to regions of the surface that had either been positively modified or were nominally neutral. Regions of modified negative surface charge effectively repelled the virus particles.

Self-diffusion of rodlike and spherical particles in a matrix of charged colloidal spheres: a comparison between fluorescence recovery after photobleaching and fluorescence correlation spectroscopy.

The fluorescence recovery after photobleaching (FRAP) method and the fluorescence correlation spectroscopy (FCS) have been applied on suspensions of highly charged colloidal spheres with a small content of rod-shaped tobacco mosaic virus (TMV) particles. Since these methods only determine the self-diffusion coefficient of the fluorescently labeled species, D(S) of the rods and the spheres could independently be measured. The ionic strength of the dispersion medium has been varied to measure self-diffusion of rods and spheres in dependence on the degree of order of the matrix spheres. In contrast to FRAP, which allows the determination of the long-time self-diffusion coefficient D(S) (L), FCS measures self-diffusion on a shorter time scale. Thus a comparison of the results that were obtained by FCS and FRAP, in combination with Brownian Dynamics simulations, gives insight into the time dependence of the self-diffusion coefficient of an interacting colloidal system. As the mean interparticle distance of the matrix is of the same order of magnitude as the length of a TMV rod, the rotational motion is influenced by the assembly of spheres around a TMV particle. Since FCS is sensitive both to translational and rotational motion, whereas FRAP, which probes the diffusion at much larger length scales, is only sensitive to the translational motion of TMV, the comparison of diffusion coefficients measured employing FRAP and FCS can give some insights in the rotational diffusion: the experimental data indicate a slowing down of the rotational motion of a TMV rod with increasing structural order of the matrix spheres.

Affinity purification of streptavidin using tobacco mosaic virus particles as purification tags

A new protein affinity purification system has been developed. Recombinant tobacco mosaic virus (TMV) was used as an affinity matrix for isolation and purification of the given protein of interest. In model experiments, streptavidin-specific heptapeptide sequence TLIAHPQ was inserted into TMV coat protein near the C end. This oligopeptide did not interfere significantly with viral replication, assembly, and movement. Recombinant TMV functioned as an epitope tag recognizing streptavidin in plant protein extracts. Plant protein extracts containing streptavidin were incubated with recombinant TMV virions. Affinity complexes of viral particles with the protein of interest were collected by centrifugation. Recombinant TMV–streptavidin complex was dissociated with 0.2 M acetic acid, pH 4.6, and was passed through membrane filter Nanosep 300 K by centrifugation. The filtrate contained pure streptavidin. Recombinant TMV was left on the filter. TMV particles collected from the filter could be used for at least two more purification cycles. The streptavidin-specific recombinant TMV system was applied successfully for purification of streptavidin from Streptomyces avidinii. The authors believe that the TMV-based affinity system can also be used for the purification of other proteins.

Dependence on sphere size of the phase behavior of mixtures of rods and spheres

By the addition of chondroitin sulfate (Chs) to the aqueous suspension of tobacco mosaic virus (TMV), the aggregation of TMV occurs at very dilute TMV concentration compared with the addition of polyethylene oxide (PEO). The difference of physical behavior between Chs and PEO is the chain conformation in solution. The Chs chain has a semirigid nature, whereas the PEO chain has a flexible nature. In this study, the Chs and PEO chains are simplified to spherical particles having different size, and we use the spherocylinder model for TMV particle. The effect of the sphere size on the phase behaviors in the mixtures of rods and spheres is investigated by Monte Carlo simulations. By the addition of small spheres, the system transforms from the miscible isotropic phase to the miscible nematic phase. On the other hand, by the addition of large spheres, the system changes from the miscible isotropic phase to the immiscible nematic phase through the immiscible isotropic phase. The different phase behaviors between the small and the large spheres originate from the difference of overlapping volume of the depletion zone. In addition, we perform the Monte Carlo simulations in the case that semirigid chains are used as the Chs chain models. The same phase behaviors are observed as the mixtures of rods and large spheres. Thus the sphere model captures the phase behaviors of rod and polymer mixture systems.

Organization of Metallic Nanoparticles Using Tobacco Mosaic Virus Templates

Self-assembled cylindrical particles of wild type and recombinant tobacco mosaic virus (TMV) were used as organic templates for the controlled deposition and organization of Pt, Au, or Ag nanoparticles. Chemical reduction of [PtCl6]2- or [AuCl4]- complexes at acidic pH gave rise to the specific decoration of the external surface of wild-type TMV rods with metallic nanoparticles less than 10 nm in size. In contrast, photochemical reduction of Ag(I) salts at pH 7 resulted in nucleation and constrained growth of discrete Ag nanoparticles aligned within the 4 nm-wide internal channel. The number of encapsulated nanoparticles increased when Ag benzoate rather than Ag nitrate was used due to reduced supersaturation associated with the lower Ag/benzoate redox couple, which enhanced the surface-templating effect of the channel wall carboxylates compared with nucleation in solution. Similar experiments using a mutant TMV with reduced negative charge along the central cavity confirmed that glutamic and aspartate acid groups were involved in site-specific deposition. Our results suggest that it should be possible to prepare 1-D arrays for a wide range of inorganic quantum dots by molecular engineering of the internal and external surfaces of self-assembled TMV tubules.

Electrophoretic Mobility of Rigid Rodlike Particles in Dilute Aqueous and Glycerol Suspensions: Comparison between Theory and Experiment

The electrophoretic mobilities of rigid rodlike tobacco mosaic virus (TMV) particles solvated in water and in 80% glycerol/20% water solutions with a wide range of electrical double-layer thicknesses have been measured by Deggelmann et al. (Deggelmann, M., Graf, C., Hagenbuchle, M., Hoss, U., Johner, C., Kramer, H., Martin, C., and Weber, R., J. Phys. Chem.98, 364–368 (1994)) and the present authors, respectively. The measured mobilities have a dependence on the electrical double-layer thickness much weaker than that predicted by theories for the mobility of cylinders of infinite length. To interpret the experiments, we develop an approximate theory for the mobility of finite aspect ratio particles with arbitrary ratios of the double-layer thickness to the rod length and diameter. This uniformly valid approximation interpolates between previous theories for infinite rod length and for infinitesimal rod thickness. A comparison of the uniformly valid theory with the experimentally measured mobilities indicates that TMV has a negative charge of 1000e in water and 400e in the glycerol solution.

Destruction of TMV and PVX caused by sap from the halo zones surrounding homologous virus-induced local lesions in leaves of host plants

When tobacco mosaic virus (TMV) and potato virus X (PVX) pre­pa­ra­tions were mixed with the sap from the halo zone (HZ) tissues surround­ing homologous virus-induced local lesions in Datura stramonium and Gomphrena globosa leaves, respectively, and the mixtures were incubated for 18 h at 37 °C, the virus particles underwent destruction. Under the electron microscope abnormal (swollen and “thin”) virions were observed in the incubated virus preparations negatively stained with phospho­tung­stic acid. Sometimes the TMV particles were “cut” across into fragments. Treatment of the virus preparations with the sap from the healthy leaves or HZ surrounding heterologous virus-induced local lesions may cause certain destructive changes of virus particles but to a far lesser extent than treatment with the sap from the homologous virus-induced HZ. Possible mechanisms of destruction of the virus particles are discussed.

The Fate of Plant Pathogens and Seeds During Anaerobic Digestion and Aerobic Composting of Source Separated Household Wastes

Anaerobic digestion is becoming a more common method for treatment of organic wastes. Little is known, however, about the effects of this process on the fate of plant pathogens and seeds. Therefore, the fates of the plant pathogens Plasmodiophora brassicae, Heterodera schachtii, Meloidogyne incognita, Ralstonia solanacearum, tobacco mosaic virus (TMV) and tomato seeds were followed during anaerobic digestion of source separated household wastes. With the exception of TMV, all test organisms were destroyed to below detectable limits within one day of anaerobic digestion at 52°C. Two days of anaerobic digestion did not reduce the concentration of TMV. However, two days of anaerobic digestion followed by 19 days of high temperature composting at 58°C reduced the concentration of infectious TMV particles by a factor of almost three orders. Anaerobic digestion followed by 12 days of composting at 68°C was even more effective. Although TMV concentrations were not eliminated entirely, we conclude that short-term high temperature anaerobic digestion followed by high temperature composting is a highly efficient process for the eradication of detrimental agents from solid wastes.

Electrochemical modification of individual nano-objects

Two examples of selective electrochemical modification of individual nanoscale objects are presented, namely the electroless metallization of a plant virus, and the coupling of substituted phenyl residues to single-wall carbon nanotubes (SWCNTs). The electroless deposition of metal was achieved selectively on the inner or outer surface of tobacco mosaic virus (TMV) particles. Covalent modification of SWCNTs, deposited on a Si/SiO 2 substrate, was performed successfully via reductive coupling of a 4-nitrobenzenediazonium salt in non-aqueous medium. An organic layer with a thickness of up to several nanometers could be deposited on isolated tubes and thin bundles.