Turnip Yellow Mosaic Virus Particles Research Articles

Empty Turnip yellow mosaic virus capsids as delivery vehicles to mammalian cells

Turnip yellow mosaic virus (TYMV) was able to enter animal cells when the spherical plant virus was conjugated with Tat, a cell penetrating peptide (CPP). Tat was chemically attached to the surface lysine residues of TYMV using hydrazone chemistry. Baby hamster kidney (BHK) cells were incubated with either unmodified or Tat-conjugated TYMV and examined by flow cytometry and confocal microscopic analyses. Tat conjugation was shown to be more efficient than Lipofectamine in allowing TYMV to enter the mammalian cells. Tat-assisted-transfection was also associated with less loss of cell viability than lipofection. Among the CPPs tested (Tat, R8, Pep-1 and Pen), it was observed that R8 and Pen were also effective while Pep-1 was not. We also examined if the internal space of TYMV can be used to load fluorescein dye as a model cargo. When TYMV is treated by freezing and thawing, the virus is known to convert into a structure with a 6–8 nm hole and release viral RNA. When the resultant pot-like particles were reacted with fluorescein-5-maleimide using interior sulfhydryl groups as conjugation sites, about 145 fluorescein molecules were added per particle. The fluorescein-loaded TYMV particles were conjugated with Tat and introduced into BHK cells, again with higher transfection efficiency compared to lipofection. Our studies demonstrate the potential of modified TYMV as an efficient system for therapeutic cargo delivery to mammalian cells.

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.

Closed-Packed Colloidal Assemblies from Icosahedral Plant Virus and Polymer

A practical process to synthesize bio-colloidal composites was developed on the basis of the noncovalent interactions of icosahedral plant virus turnip yellow mosaic virus (TYMV) and poly(4-vinylpyridine) (P4VP). TYMV particles fully covered the surface of a P4VP ball with a hexagon-like packing. The raspberry-like morphology of TYMV-P4VP colloids and the packing pattern of TYMV were revealed by transmission electron microscopy (TEM), field-emission scanning electron microscopy (FESEM), dynamic light scattering (DLS), and synchrotron small-angle X-ray scattering (SAXS). The size of TYMV-P4VP colloids was controlled readily by varying the mass ratio of virus and polymer. A simplified model was established to explain the experimental data.

Stability of Belladonna Mottle Virus Particles: the Role of Polyamines and Calcium

Summary The stability of belladonna mottle virus (BDMV) has been studied with respect to elevated pH and to freezing and thawing. BDMV, purified by a modified procedure, was stable at alkaline pH, in contradiction to earlier reports. This difference in the stability could be attributed to the presence of 90 to 140 molecules of spermidine, 20 to 50 molecules of putrescine and 500 to 900 calcium ions in each virus particle. The polyamines could be easily exchanged with other cations such as potassium or caesium and this resulted in a loss of particle stability. These cations may therefore play a role in maintaining the integrity of particle structure. The formation of empty protein shells as a result of freezing and thawing BDMV particles parallels earlier observations on turnip yellow mosaic virus particles.

Studies on the Phenomenon of Turnip Yellow Mosaic Virus RNA Release by Freezing and Thawing

Summary In particles of turnip yellow mosaic virus (TYMV), the interactions between proteins are particularly strong when compared to those between proteins in some other icosahedral viruses. Intact RNA is released from TYMV particles on freezing and this process has been studied by examining several parameters that influence cryodenaturation such as dehydration, pressure, aggregation and the presence of protective agents (glycerol and ammonium sulphate). Pressure of 1.5 × 108 Pa had no effect on virus particles whereas dehydration of a virus suspension had a drastic effect. Aggregate formation resulting from freezing of solutions containing high virus concentrations seems to be a prerequisite for RNA release; cryoprotective agents hindered RNA release.

Cell surface labelling of mononuclear cells with antisera associated to turnip yellow mosaic virus or alphalpha mosaic virus particles. A freeze-etch study

Turnip yellow mosaic virus (TYMV) and alphalpha mosaic virus (AMV) were used as immuno-electron microscopical markers to detect cell surface receptors on mononuclear cells in freeze-etch replicas. TYMV particles were conjugated with vacuum-distilled glutaraldehyde to rabbit IgG anti-mouse immunoglobulins (TYMV-RAMIg conjugate) or to rabbit IgG anti-mouse theta antigen (TYMV-RAMTh conjugate). B-lymphocytes incubated with TYMV-RAMIg conjugate showed either randomly distributed particles or patches of virus particles on the etched surface of the cell membrane. Mouse thymocytes incubated with TYMV-RAMTh conjugate, however, showed only a random distribution of the virus particles. Human mononuclear cells incubated with rabbit IgG anti-AMV and AMV for the demonstration of the receptors for the Fc fragment of IgG showed the oblong shape of the AMV particles on the etched cell membrane, Fc receptors were either randomly distributed or aggregrated into patches. It is concluded that both types of virus particles are useful markers for the demonstration of membrane receptors in freeze-etch replicas of labelled cells.

Sites of coat protein accumulation in turnip yellow mosaic virus-infected cells

Empty viral protein shells or viral antigen have been identified in the nuclei of Chinese cabbage infected with turnip yellow mosaic virus, Nicotiana glutinosa infected with eggplant mosaic virus, cucumber infected with okra mosaic virus, and bean infected with Desmodium yellow mottle virus. For turnip yellow mosaic virus, viral coat protein can be detected from an early stage of infection at two sites: (i) in the nucleus, where coat protein in the form of empty protein shells accumulates, and (ii) in the electron-lucent zones in the cytoplasm over the clustered vesicles of chloroplasts. We conclude that this electron-lucent material contains pentamers and hexamers of coat protein because particles seen within it on freeze-fracturing have the right dimensions, and because the material reacts with antibody directed against intact TYMV. A mechanism is proposed by which turnip yellow mosaic virus particles are assembled at the necks of the peripheral vesicles from an emerging strand of RNA combining with pentamers and hexamers of coat protein lying in an oriented fashion within the lipid bilayer of the outer chloroplast membrane.

Infection of brassica leaf protoplasts by turnip yellow mosaic virus

Summary. Conditions optimal for the infection of Brassica leaf protoplasts by turnip yellow mosaic virus (TYMV) were found to be: pH 5.4-5.8 with citrate buffer; concentration of poly-L-ornithine, Izg/ml; concentration of TYMV, 0.2-1.0 lzg/ml. More than 90% of Brassica rapa and B. sinensis protoplasts were infected under these conditions. TYMV replication in Brassica protoplasts was followed by quantitating the virus extracted from protoplasts and separated by sucrose density gradient centrifugation. Brassica protoplasts produced I to 2 × 106 TYMV particles per cell within 48 hrs. Infection by TYMV induced the formation of polyplasts (aggregates of chloroplasts) in Brassica protoplasts. Polyplast formation paralleled the appearance of TYMV-specific immunofluorescence and could thus be used conveniently to determine the number of infected protoplasts. TYMV replication in Brassica protoplasts was resistant to actinomycin D and chloramphenicol, but was completely inhibited by cycloheximide.

Crystallization of turnip yellow mosaic virus in wilted leaves of Chinese cabbage by treatment at high temperature.

Electron microscopic examination of Chinese cabbage leaves systemically infected with turnip yellow mosaic virus (TYMV) showed that numerous particles assumed to be virus were present in the cytoplasmic spaces between clumped chloroplasts. In the general ground cytoplasm TYMV particles were undistinguishable from the ribosomes. When whole leaves, detached from intact plants showing mosaic, were heated at 60 C for 5 min and kept at room temperature (about 20 C) for 18 hr, particles located in the spaces between chloroplasts and general cytoplasm were apparently crystallized. No such crystalline arrays were seen in healthy cells. The interparticle distance in the crystals formed in wilted tissue was 270–300 A. There was no evidence that cytoplasmic ribosomes have a tendency to crystallize under such conditions. It is suggested that TYMV particles are crystallized by the wilting procedure used.

Interactions of mercuric chloride with turnip yellow mosaic virus: Dissociation of the virus and reassociation of the reaction products

The action of HgCl 2 on turnip yellow mosaic virus (TYMV) has been investigated by analytical ultracentrifugation and electron microscopy, as well as by sucrose gradient analysis of 32P -or 35S-labeled virus. As increasing amounts of HgCl 2 (from r = 0.15 to r = 0.6, where r is the molar ratio of added HgCl 2 to nucleotide phosphorus) were added to TYMV, the virions were dissociated into RNA-free protein and a nucleoprotein containing an amount of protein increasing with r. At r > 0.9, Hg(II) bound to TYMV without disruption of the virion. These nucleoproteins were dissociated upon addition of mercaptoethanol. At r = 0.15, in the presence of chloride ions, Hg(II) bound only to the -SH groups of TMYV or artificial top component (ATC), leading to the total disruption of the protein shell. In the absence of chloride ions only a slight degradation was observed. When an adequate amount of HgCl 2 was added in two steps ( r = 0 → 0.45 → 0.9 or 1.5), the protein reassociated with the nucleoprotein moiety into particles which were less regular in shape and size than native TYMV. The nature of the forces stabilizing native and mercurated TYMV particles as well as ATC are discussed.

Electron microscope observations on the structure of the protein shell of turnip yellow mosaic virus

High resolution and high magnification electron micrographs of turnip yellow mosaic virus particles prepared by the negative staining procedure and the rotation technique are presented. Excellent agreement is found between these micrographs and a rhombic triacontahedron model with 32 subunits arranged in an icosahedral pattern with 5∶3∶2 symmetry. The protein shell of the virus is constructed of 32 morphological subunits. A discussion and interpretation of the electron microscope observations and the physico-chemical data is provided. All the evidence so far is strongly suggestive of 180 structural units representing possibly the total number of peptides in the virus protein shell. Some of the artifacts of the roration technique are also discussed.

Electron microscope observations on the structure of turnip yellow mosaic virus

Electron micrographs of turnip yellow mosaic virus particles prepared by the negative staining technique show clearly defined internal structure, which indicates that the protein shell of this virus is constructed of 32 subunits in an icosahedral arrangement. The structure inferred from the electron micrographs satisfies the X-ray data, and contains subunits which occupy two structurally distinct sites.