Tobacco Mosaic Virus Preparations Research Articles

Increased Efficiency of Radiation Inactivation of Virions by Gold Nanoparticles

AbstractGold nanoparticles (GNPs) are promising radiosensitizers for cancer radiotherapy. Moreover, they can be used in the same way for radiation processing and sterilization. Such application of GNPs is of practical interest since it may significantly reduce the dose load and expand the application of radiation treatment. In the present study, the high radiosensitization effect of GNPs in relation to viral particles is demonstrated for the first time. The preparations of tobacco mosaic virus (TMV) are used as an experimental model, insofar as this virus has the same properties as animal and human ones but is safe for humans. Irradiation with 45 kVp X‐ray to the doses of 4 and 7 kGy leads to a decrease in the infectious activity of TMV virions up to 1.92‐ and 2.70‐fold, respectively. At the same time, irradiation in the presence of 0.4 mg mL−1 of 12 nm spherical GNPs increases the efficiency of virus inactivation up to 15‐ and 22‐fold. The GNPs enhance both the damage to capsid protein due to the enhanced generation of reactive oxygen species and genome RNA due to the emission of secondary radiation. These results show the great prospects of the application of high‐Z nanoparticles in radiation treatment.

Elimination of Tobacco mosaic virus from irrigation runoff using slow sand filtration

Slow sand filters (SSF) are effective in removing pythiacious organisms from captured runoff to prepare the water for reuse in irrigation. Plant viruses can also be present in this runoff, but until this study, it hadn’t been clearly shown that SSFs can remove these pathogens. In this study, purified preparations of Tobacco mosaic virus (TMV) were regularly added to captured irrigation runoff water that was provided to the SSFs. Water samples were collected weekly from sampling ports above and below the sand bed and tested for the presence of TMV by ELISA and inoculation to indicator plants. Biologically viable TMV passed through the sand filters for approximately 5 weeks, followed by a gradual reduction and then elimination of detectable amounts of the virus by week 6–9. Testing of water samples continued for several more weeks after the initial loss of virus titer to confirm complete elimination. This is the first controlled study showing elimination of a plant virus from captured irrigation runoff using SSFs. Since TMV is a highly robust organism, it is likely that other plant pathogenic viruses can also be removed using SSF systems.

Cotranslational disassembly of tobacco mosaic virus in vitro

Heterogeneous nucleoprotein preparations of tobacco mosaic virus (TMV), stored at pH 7.0, were treated briefly at pH 8 and then incubated in mRNA-dependent rabbit reticulocyte lysate. The treatment at pH 8.0, under conditions which did not cause detectable polar disassembly as judged by exposure to micrococcal nuclease, caused TMV to stimulate in vitro translation up to 100-fold over background. High-molecular-weight products, characteristic of TMV, appeared, albeit at a significantly slower rate than when naked TMV RNA was used as template. Ribonucleoprotein material from cycloheximide-treated incubations was examined in the electron microscope to reveal a subpopulation of rodlets (approx 5%) with clusters of ribosomes at their concave 5′ ends. It is proposed that pH 8 treatment removes the last turn of protective coat protein subunits from the rods and “exposes” at least a 49-nucleotide segment of the efficient 5′-leader sequence of TMV RNA for interaction with 40 S ribosomal subunits. Ribosome translocation during protein synthesis presumably dislodges further coat protein subunits progressively while continuing protection of the incoming TMV RNA.

Chloroplast DNA transcripts are encapsidated by tobacco mosaic virus coat protein

Preparations of tobacco mosaic virus contain pseudovirions, particles resembling virions but containing host rather than viral RNA. The encapsidated host RNA was found to be composed of discrete-sized species derived from a large portion of the chloroplast genome except that very little, if any, ribosomal RNA is present. Pseudovirions contain the same chloroplast DNA transcripts as those detected in extracts from uninfected leaves, although not always in the same relative amounts. Several strains of tobacco mosaic virus were tested and all were found to contain pseudovirions, with the U2 strain containing more than the others.

Characterization and in vitro translation of the RNAs from less-than-full-length, virus-related, nucleoprotein rods present in tobacco mosaic virus preparations

Cells of plants infected with any of three strains of tobacco mosaic virus (the cowpea, common, and a wheat strain) contain, in addition to full-length virus rods, a heterodisperse population of rod fragments. RNA extracted from purified virus showed that some of these rods were of discrete sizes; the RNAs ranged in molecular weight from about 0.28 to 1.7 × 10 6, containing 3′-ends identical to the original 3′-end of viral RNA, as judged by their capacities to bind a specific amino acid. Only the cowpea strain produced a rod containing a small (0.28 × 10 6 M r) RNA, which is known to be the mRNA for coat protein. Each strain produced an RNA of 0.68 × 10 6 M r (intermediate-length RNA) which coded in vitro (using a wheat germ system) for an ∼30,000 M r polypeptide. Other RNAs (0.9 to 1.7 × 10 6 M r) were less similar in molecular weights among the three strains, but the predominant in vitro product of each was also the ∼30,000 M r polypeptide. peptide maps comparing the translation products of the short and intermediate RNAs of the cowpea strain showed they were distinct polypeptides. We conclude that at least some of the less-than-full-length viral rods found in preparations of the common and wheat strains of TMV, as well as those previously reported for the cowpea strain, represent the encapsidation of subgenomic portions of the viral RNA which are engendered during viral replication.

The application of TMV particles as an internal magnification standard for determining virus particle sizes with the electron microscope

At various sites on three grids with crude-sap preparations of tobacco mosaic virus (TMV), electron micrographs were made and per photograph 50–200 particles were measured. Calculated average lengths turned out to differ up to 4% according to the site photographed. Calculated lengths appeared to depend on potentiometer settings required for focussing. This is due to the effect of preparation height or object position along the optical axis of the electron microscope on image magnification. This is further supported by the effect of additional rings underneath the grid and of artificial bending of the grid. Because it is hard to reproduce exactly electron microscope magnification, and at given potentiometer settings per photograph calculated average lengths may still vary up to c. 4%, the application of internal standards is essential for determining the size of unknown viruses. With TMV only relative particle sizes can be determined, and even with more accurate non-biological standards no absolute virus sizes will be found.

The Donnan effect in tobacco mosaic virus and its components

Osmotic pressure studies were carried on tobacco mosaic virus (TMV) and its components, protein and RNA, as well as on bis(3,3′-aminopropyl)amine, reported to be present in TMV preparations. Solvents were phosphate and barbital buffers at different values of pH and ionic strength. Measurements were made at room temperature. The Donnan effect was exhibited by TMV protein in phosphate buffer of 0.01 ionic strength at pH values ranging between 5.8 and 7.5. The observed values of the Donnan effect at pH 5.8 and 5.97 were in reasonable agreement with theoretical values calculated from the charge obtained by hydrogen ion titration. TMV-RNA in phosphate buffer at pH 7.5 and ionic strength 0.01 did not exhibit more than 1% of the expected Donnan effect. This is explained tentatively as the result of firm binding of metal ions. Negative values of osmotic pressure were observed with bis(3,3′-aminopropyl)amine. Similar anomalous osmosis was sometimes observed with TMV protein and with TMV. In agreement with earlier observations, TMV did not exhibit the Donnan effect in phosphate buffer of 0.01 ionic strength at pH values ranging from 5.5 to 8.0. However, TMV dialysed extensively in the presence of EDTA at pH 8.5 and TMV produced by reconstitution of purified protein and RNA did exhibit the Donnan effect in both phosphate and barbital buffers. The magnitude was of the same order as that calculated from the net charge determined by hydrogen ion titration. When reconstituted TMV, which did exhibit Donnan effect, was treated with calcium ions, the effect was abolished.

Plant Viruses with a Divided Genome

A considerable number of plant viruses produce in infected plants more than one nucleoprotein component. Typical examples are tobacco rattle virus (TRV) , alfalfa mosaic virus (AMV), viruses of the cowpea mosaic virus (CPMV) group, tobacco streak virus (TSV) and brome mosaic virus (BMV). It has been shown in recent years for some of these muItiparticulate viruses that the genetic information necessary for virus multiplication is di­ vided among two or more nucleoprotein components. Discovery of plant viruses with a divided genome has changed the con­ cept of the nature of viruses. The classical concept implied that the mature virus, the ultimate phase of viral development, was a single particle, the vir­ ion, containing the genome of the virus. This is of course still true for many plant viruses, but no longer for all. Several important groups of plant viruses have been recognized to have a genome divided among two or more physi­ cally separable particles. A certain heterogeneity occurs in practically all purified preparations of plant viruses. In preparations of tobacco mosaic virus (TMV) for example, rodshaped particles with a modal length of 300 m,u predominate, but smaller and longer rods occur also. The presence of such particles has been explained by possible fragmentation of 300 mft particles during purification, faulty syn­ thesis of particles, or, in case of longer particles, by aggregation. Such hetero­ geneity is quite normal in preparations of rodshaped and threadlike viruses. Similarly, preparations of spherical viruses may contain some particles with fragmented RNA. This type of particle we shall regard as defective; it con­ sists of viral structural proteins and fragments of the viral genome. It is possi­ ble that these defective particles have some biological significance, and that they can interfere with virus replication. The infectivity of TMV, for exam­ ple, is enhanced by the presence of short particles derived from the virus in the inoculum (23, 37). In these cases it is a question of occurrence of defec­ tive particles, besides nondefective homOlogous virus particles which contain a complete genome necessary for multiplication (36). Heterogeneity in such virus preparations does not reflect functional heterogeneity. This is different from the multiparticulate viruses we will discuss here. Plant viruses with a divided genome are characterized by physical hetero-

Some factors affecting particle length distribution in tobacco mosaic virus preparations

The length of tobacco mosaic virus (TMV) particles in crude extracts of infected tobacco leaves and in preparations purified by ultracentrifugation and by precipitation with ammonium sulfate have been determined by electron microscopy. Preparations with high proportions of particles longer than 300 mμ were prepared by treating virus preparations with acetate buffer, pH 4.8. Preparations with high proportions of particles shorter than 300 mμ were obtained when phosphate buffer, pH 7.2, was used. The particle-length distribution in these preparations was readily reversed by dialysis against the appropriate buffer for 3 days at 4 °. TMV preparations purified by ammonium sulfate precipitation or by ultracentrifugation and resuspending pellets in buffer at pH 4.8 or 7.2, all had similar infectivity in spite of their different particle-length distributions. Virus purified by ammonium sulfate precipitation, when dialyzed against phosphate buffer, pH 7.2, contained an increased proportion of particles shorter than 300 mμ yet its infectivity increased slightly. It is suggested that particles shorter than 300 mμ may enhance the infectivity of particles 300 mμ or longer.

Electron microscopy of partially degraded tobacco mosaic virus

Electron microscopy of purified preparations of tobacco mosaic virus (TMV) that had been treated with phenol revealed particles from which the protein had been stripped from one end only, particles from which the protein had been removed from both ends, particles from which protein had been removed from one or more places in the middle of the rod, and still other particles from which no protein at all had been removed. It appears, therefore, that degradation of TMV by phenol occurs in a random fashion along the virus rod. Numerous long ribonucleic acid threads were found in all preparations. Some of the partially degraded virus particles were approximately 3000 A long and 200 A wide. The exposed nucleic acid of such particles was approximately 66 A wide. Partially degraded preparations were about 1 200 as infectious as the intact virus, and when such preparations were incubated with ribonuclease the infectivity was almost completely lost.

The accumulation in plants of chromatographically separated components of tobacco mosaic virus

Chemically purified tobacco mosaic virus preparations were separated into several components by stepwise elution of a cation-exchange resin (Amberlite IR-120) and ECTEOLA cellulose columns. The amounts of the components in preparations from source plants infected for different periods was determined. Two of the components separated by ECTEOLA cellulose chromatography started to accumulate a day later than the others.

PENETRATION OF STAIN IN ULTRATHIN SECTIONS OF TOBACCO MOSAIC VIRUS.

Purified preparations of tobacco mosaic virus (TMV) and pieces of tomato leaves infected with TMV were embedded in methacrylate or epoxy resin, sectioned, and stained with 1.0% strontium permanganate for electron microscopy. In sections containing purified and intracellular virus, the apparent length of stained particles varied directly with section thickness, indicating stain penetration beyond the surface of the section. Penetration was demonstrated also by stereoscopy. Penetration was less complete when sections were allowed to dry before staining. In most instances the number of identifiable particles per unit area was independent of section thickness but increased when both surfaces of the sections were stained instead of only one surface. Staining was prevented by thin films of methacrylate or epoxy resin placed between the virus section and staining solution. Most results supported the view that electron scattering capacity was enhanced only in particles which intersected the surface of the section...

ON THE RIBONUCLEASE ACTIVITY ASSOCIATED WITH TOBACCO MOSAIC VIRUS PREPARATIONS.

Abstract A very sensitive method for the detection of ribonuclease activity in tobacco mosaic virus (TMV) preparations is described. All TMV preparations that have been examined exhibit ribonuclease activity, the level of which depends on the method used for purifying the virus. Protein isolated from the virus also shows this activity. The ribonuclease has a pH optimum in the range 8.5–9.0, and its activity is not dependent on the presence of either magnesium or pyrophosphate. Attempts to remove the enzyme from TMV by additional steps of purification were partly successful. A nucleoprotein pellet prepared from healthy tobacco leaves by a procedure similar to that used for the preparation of TMV from infected leaves exhibits ribonuclease activity that has the same pH optimum as that of TMV preparations. It is concluded that the ribonuclease in TMV is not an integral part of the virus, but is probably due to the presence of host-plant particles, such as ribosomes, which are not completely removed during the isolation of the virus.

A polarographic method for the detection of protein impurities in preparations of tobacco mosaic virus

A polarographic method for the detection of protein impurities in preparations of tobacco mosaic virus

A Preliminary Study of Tobacco Mosaic Virus by the Gel Diffusion Precipitin Tests

SUMMARY: A number of proteins can be separated from purified preparations of tobacco mosaic virus; they differ from each other antigenically and all differ from the intact virus by not possessing all antigenic determinant groups possessed by the virus. Some of these proteins are easily detached from the virus by such mild treatments as placing it in a protein solution or in agar gel. These are antigenically identical with the ‘X-protein’ which remains in the supernatant fluid when the virus is sedimented by ultracentrifugation from sap of infected plants. More of these proteins are detached from the virus by placing it in borate buffer at c. pH 8·7. When a more drastic treatment is applied, such as incubation at c. pH 10, which disintegrates a proportion of the virus, still more of these proteins are released, but then some proteins antigenically different from those of ‘X-protein’ are also released.

On the Tobacco Mosaic Virus contained in Manufactured Tobacco.

The present paper deals with a study on tobacco mosaic virus (TMV) contained in manufactured tobacco. The following kinds of commercial tobacco which had been manufactured in Japan in 1954, were tested in order to see to what extent they might carry the TMV: 1) six kinds of cigarettes and their scraps, 2) one kind of cigars, 3) two kinds of pipe obaccos, 4) three kinds of fine cut tobaccos and their scraps. The samples were collected from 21 factories all over the country, and were tested by rubbing inoculation of tobacco seedlings with extracted juice with carborundum. Of 126, 126, 3, 6, 42 and 42 samples tested, 123 (97%) of cigarettes, 124 (98%) of their scraps, 3 (100%) of cigars, 3 (50%) of pipe tobaccos, 36 (86%) of fine cuts, and 41 (98%) of their scraps, respectively, caused mosaic on inoculated tobacco plants. None of samples of Momoyama, pipe tobacco, caused mosaic. However, the yellow component separated from the sample caused mosaic, though the blackened one did not. According to the tests of local lesion method on Nicotiana glutinosa, there were no consistent differences in TMV concentration between cigarette and fine cut, and between each of them and its scrap. The TMV concentrations examined in 3 cigarette samples were about 5×10-4-10-4 of that in fresh mosaic leaf grown in a greenhouse, per same weight. These figures are rather high, regarding the danger of infection of tobacco plant in the field. Electron micrographs of three preparations of TMV purified by ultracentrifuge from distilled water extracts of cigarette, from tobacco plants inoculated with the extracts, and from plants inoculated with the ordinary strain of TMV (a strain stocked in our laboratory as an ordinary one), show that the length distribution of particles is generally similar, but there was some indication that the former two preparations contained more short particles than the last one.

Some components of tobacco mosaic virus preparations made in different ways.

Some components of tobacco mosaic virus preparations made in different ways.

Deuteron bombardment of oriented tobacco mosaic virus preparations.

Deuteron bombardment of oriented tobacco mosaic virus preparations.

Effects of X-ray radiation upon the pathogenicity and antigenicity of tobacco mosaic virus

The present work was undertaken in order to investigate the effects of X-ray radiation upon the pathogenicity and antigenicity of tobacco mosaic virus (TMV).The virus preparation used in this work was the aqueous suspension of TMV purified by Bawden's procedure. The antiserum was prepared by injecting rabbits with the purified TMV. The infectivity of the irradiated virus was tested by the half-leaf method using Nicotiana glutinosa as assay plants. In serological tests of TMV, the precipitin reaction was used. The virus preparation 2 or 3mm thick in a Petri dish of 5cm in diameter was irradiated at 5cm distance from an anticathode of an X-ray tube run at 60KVP and or 10mA. The mean wave-length of X-ray used in this work was 0.4A, and dosages measured were 2400r/min. in the case of 15mA and 1600r/min. in that of 10mA. The pH of the virus preparations were 6.0∼6.2 and their temperatures were 14°∼20°C during the irradiations, and as the X-ray tube employed was water-cooled no rising of temperature of the preparations due to the irradiations was recognized.The results of experiments are summarized as follows:(1) The non-infective preparations of TMV which had been inactivated by X-ray radiation always showed to be serologically active, and it was moreover found that the serological activities of irradiated preparations increased within the limits of dosage used in the experiments, on the contrary of decreasing of the infectivity (Tables 1 and 2).(2) The logarithm of the surviving fraction has been plotted against the dosage of irradiation according to the results of the virus activity assay (half-leaf mnethod), and the survival curve was shown by a straight line (Fig. 1 and 2). Consequently, these results seem to agree with the view that the inactivation of virus by ionizing radiation is caused by a single ionization or a single cluster of ionization, as the inactivation follows a simple exponential curve.(3) The inactivation doses (the 37% doses) were estimated from the above-mentioned curves, to be approximately 28×104r in the case of preparations of 0.2% and 2×104r in the occasion of 0.02%.

The behaviour of some plant viruses after exposure to ultraviolet radiation.

SUMMARY: Preparations of tobacco mosaic virus (TMV) inactivated by ultraviolet radiation interfered slightly with infection by active tomato bushy stunt (BSV) and Rothamsted tobacco necrosis (RTNV) viruses, and much more so with active TMV. Similarly, inactivated RTNV interfered slightly with infection by TMV and more so with active RTNV. In contrast, inactivated BSV did not affect the numbers of lesions produced by active virus preparations. The residual infectivity of irradiated preparations of RTNV and BSV was greater when inoculated plants were exposed to light than when they were kept in the dark. This occurs because of some light-sensitive mechanism in the host cells, and exposing the irradiated virus preparations to visible light did not affect their infectivity. Irradiated preparations of TMV had the same residual infectivity whether plants were placed in the light or dark after inoculation. Although the three viruses have particles of different sizes and shapes, the course of inactivation by ultraviolet with each approximated closely to that of a first-order reaction.