Tobacco Mosaic Virus Mutant Research Articles

Characterization of a temperature-sensitive mutant of tobacco mosaic virus deficient in synthesis of all RNA species

The biochemical lesion of the temperature-sensitive, replication-deficient mutant of tobacco mosaic virus (TMV), III 2-35, was examined in temperature shift experiments. Following a shift-up to the restrictive temperature (35°), incorporation of 32P into viral single-stranded, replicative form, and replicative intermediate RNAs immediately ceased. Following the cessation of synthesis at 35°, III 2-35 RNA synthesis immediately resumed when infected leaves were shifted back to 25°, suggesting that the activity of the viral RNA replicating complex is temperature sensitive in vivo. However, the in vitro activity of the membrane-bound replicase was not temperature sensitive, although its optimum temperature profile and stability at 35° were different from that of the wild type enzyme.

Stability of mutations conferring temperature sensitivity on tobacco mosaic virus.

Among several temperature-sensitive mutants of tobacco mosaic virus, reversion to wild-type phenotype is an event sufficiently rare that essentially pure stocks of mutant virus can easily be maintained. The temperature-sensitive phenotype of many of these mutants is stringently expressed. Our data show that most of our mutants are suitable for use in biochemical experiments.

Fluorescence study of tobacco mosaic virus protein

Protein fluorescence properties of tobacco mosaic virus [3 Trp residues per monomer (positions 17, 52, 152)] and of two tobacco mosaic virus mutants [green tomato atypical mosaic virus, 2 Trp (52, 152) and cucumber virus 4, 1 Trp (unknown position)] have been studied. Emission spectra, fluorescence quantum yields and lifetimes were determined. Results showed that protein fluorescence is due to buried Trp only, except for the cucumber virus 4 strain, in which Tyr also contributes to the emission. Comparison of the three strains showed that Trp 17 and Trp 52 have high fluorescence yields ( ϕ 17 = 0.29; ϕ 52 = 0.37) whereas Trp 152 (probably present in cucumber virus 4) is strongly quenched ( ϕ 152 = 0.035). An unusually efficient Ayr → Trp energy transfer was observed in tobacco mosaic virus protein, indicating that most of four Tyr residues are located near the highly fluorescent Trp.

The state of the tobacco mosaic virus protein subunit at the dropping mercury electrode

The conformation—dependent masked state of the —SH group of the single cysteinyl residue present in the polypeptide chain of the tobacco mosaic virus (strain Vulgare) protein subunit remains preserved at the dropping mercury electrode in ammoniacal buffer (pH 9.4) containing Co(III) ions (Brdiča's solution) at 0 °C. This result has been obtained by parallel d.c. polarographic analyses and sulfhydryl tritrations in the course of the reversible denaturation of the tobacco mosaic virus protein complemented by polarographic analyses of the proteins of two tobacco mosaic virus mutants. The results obtained speek against the view that in general, the protein molecules unfold at the surface of the mercury electrode.

A procedure for specifically selecting temperature sensitive mutants of tobacco mosaic virus

A procedure is described that permits the selection of functionally deficient, temperature sensitive mutants of tobacco mosaic virus based upon the time of local lesion formation on the appropriate host plant.

Selection of temperature-sensitive mutants of tobacco mosaic virus by lesion morphology

Two mutants of tobacco mosaic virus (TMV) were isolated, based on their inability to produce “halos” around local lesions in plants that, 3 days after inoculation, had been placed at 35° for 1 day. The two mutants differed from wild type and from each other in the rate of lesion enlargement at 23°, in systemic production of virus at 35°, and in survival during a 12-hr period at 35° administered during the early stages of infection. Although the characteristic by which these mutants were isolated had been identified previously with non-coat-protein temperature-sensitive mutants of TMV, both of the mutants exhibited coat-protein alterations, and systemic growth of one of the mutants was detected at 35°.

The amino acid sequence of the cowpea strain of tobacco mosaic virus protein

The amino acid sequence of the coat protein of the cowpea strain of tobacco mosaic virus (cowpea virus) has been determined. The tryptic peptide overlaps were obtained by digesting the protein with chymotrypsin and separating and analysing the lysine- and arginine-containing chymotryptic peptides. The primary structure of cowpea virus protein has been found to differ markedly from that of any other known strain of tobacco mosaic virus, and contains 3 amino acid residues more and 96 amino acid changes from the type strain. The significance of the distribution of those areas of the protein in which the amino acid residues are the same for all naturally occurring strains and chemically induced mutants of tobacco mosaic virus so far studied and the residues that form the important carboxyl · carboxylate pairs are discussed.

The cell-free synthesis and assembly of viral specific polypeptides into TMV particles

In a cell-free system derived from wheat germ, the ribonucleic acid of the tobacco mosaic virus mutant Ni 568 directs the synthesis of polypeptides with molecular weights from 10,000 to 140,000, but there is little synthesis of a product equal in size to the coat protein. However, further incubation of the cell-free products with or without extracts from tobacco leaves resulted in the appearance of 35S methionine labeled polypeptides comigrating with tobacco mosaic virus coat protein on sodium dodecyl sulfate-acrylamide gels. Tobacco mosaic virus particles assembled in the presence of these treated cell-free products were shown to be radioactive and co-banded with authentic tobacco mosaic virus during equilibrium centrifugation in cesium chloride. Upon dissociation the assembled particles were found to contain several labeled polypeptides, the most prominent of which comigrated with tobacco mosaic virus coat protein on sodium dodecyl sulfate-acrylamide gels. Furthermore tryptic digests of these polypeptides contained a 35S-methionine labeled peptide with a mobility during high voltage paper ionophoresis at pH 6.5 precisely coincident with that of the only methionine-containing peptide in the coat protein.

Protein synthesis in virus-infected plants: III. Effects of tobacco mosaic virus mutants on protein synthesis in Nicotiana tabacum

Nicotiana tabacum, infected with three widely differing chemically produced and one natural mutant of tobacco mosaic virus (TMV), was pulse labeled with [ 3H]leucine. Uninfected plants were similarly pulse labeled with [ 14C]leucine. The 3H- and 14C-labeled leaf tissues were combined, fractionated into subcellular fractions, denatured, dialyzed, and electrophoresed on polyacrylamide gels in SDS. Increases or decreases in the 3 H 14 C ratio in gel slices indicated changes in the proteins synthesized in the infected fractions as compared to the uninfected control fractions. In contrast to wild-type TMV, where only coat protein was detected ( Singer, 1971), protein synthesis differed markedly among the mutants and their subcellular fractions in both the number and molecular weights of the protein peaks. No protein was found to be common to all strains in the same subcellular fractions except the coat protein. In addition, decreases in the 3 H 14 C ratio were found, indicative of suppression of specific host proteins. The total molecular weights of proteins appearing upon infection with some mutants exceeded the coding capacity of the viral nucleic acid, and there was no indication that any of the very large proteins found were precursors. It appears probable that these multiple responses specifically produced by mutants are the result of changes in host protein synthesis caused by the infection.

Biological and physico-chemical properties of an atypical mutant of tobacco mosaic virus.

A mutant of tobacco mosaic virus (TMV) was isolated from a population of TMV-RNA molecules inactivated to 0.1% level of survival by treatment with nitrous acid This mutant is poorly transported in “Samsun” tobacco and induces chlorotic spots, oak-leaf patterns, ringspots and necrosis. Rate zonal sucrose density-gradient and equilibrium sedimentations in sucrose and CsCl indicate that the purified mutant preparations contain a heterogenous population of particles including some “free” coat protein. These vary in length, but approximately 5–10% are infectious rods of standard (15×300 nm) dimensions. Purified mutant RNA contain an overabundance of fragmented and a few infectious molecules. Compared to the parent strain, the mutant capsid protein contains two additional residues of arginine and one of glycine, but lacks one residue each of glutamic acid (or glutamine), serine and threonine. These observations and other data based on temperature sensitivity, exposure to urea and serology indicate that the mutant particles are inherently unstable and break during the purification procedure.

The reactivity of functional groups as a probe for investigating the topography of tobacco mosaic virus. The use of mutants with additional lysine residues in the coat protein.

Several mutants of tobacco mosaic virus that contain additional lysine residues as a result of mutations in the coat protein were investigated. Mutant E66 has a lysine residue replacing asparagine at position 140 when compared with the wild-type vulgare and this lysine residue reacts readily in the intact virus with methyl picolinimidate. Mutant B13a has two new lysine residues in the coat protein, replacing a glutamine at position 9 and an asparagine at position 33, whereas mutant B13b has the single replacement of glutamine by lysine at position 9. The lysine residue at position 9 in mutants B13a and B13b also reacts readily with methyl picolinimidate in the intact virus but the lysine at position 33 in mutant B13a did not react under these conditions. However, when the isolated coat protein from mutant B13a was treated with methyl picolinimidate, the lysine residue at position 33 did become modified, showing that the loss in reactivity of this residue towards the imidoester in the intact virus is a result of the assembly of the protein subunit into the virus structure. These results are compatible with and extend previous studies on the sero-logical properties of mutants of tobacco mosaic virus and illustrate the value of methyl picolinimidate as a reagent for probing the accessibility of amino groups in proteins. When intact tobacco mosaic virus (vulgare) was treated with p-iodobenzenesulphonyl chloride, no reaction with the lysine residues at positions 33 or 68 in the virus subunit could be detected but complete modification of tyrosine-139 was achieved. This result also extends previous studies with other reagents. The usefulness of the differential reactivity of the lysine residues in tobacco mosaic virus and its mutants as a means of attaching heavy-atom labels at chemically defined positions for subsequent X-ray-diffraction analysis and the implications of these experiments for deciphering the folding of the peptide chain in the virus subunit are discussed.

Host Range and Purification of the Nucleic Acid of a Defective Mutant of Tobacco Mosaic Virus

Host Range and Purification of the Nucleic Acid of a Defective Mutant of Tobacco Mosaic Virus

M II-16, an artificial symptomless mutant of tobacco mosaic virus for seedling inoculation of tomato crops

De bruikbaarheid van een door inwerking van salpeterigzuur verkregen, symptoomloze mutant van het tabaksmozaiekvirus (TMV) is gebleken uit een volledige bescherming van planten tegen een infectie met andere stammen van het virus en uit bevredigende proefresultaten onder praktijkomstandigheden. Het probleem doet zich voor dat de mutant in feite deel uitmaakt van een stammenmengsel, waarbinnen de verhoudingen echter ten gunste van de mutant zijn te beinvloeden.

Circular dichroism studies on coat proteins of some strains and mutants of tobacco mosaic virus

1. 1. Tobacco mosaic virus (TMV) protein has in near ultraviolet a complex but well resolved circular dichroism (CD) spectrum at room temperature. There are seven positive bands at 248, 252, 257, 265, 274, 281 and 291 nm, and a negative one at 296 nm. The CD spectrum is pH-dependent. The shape of the pH-dependence curves and the comparison with CD spectra of model compounds suggest that the bands at 248, 252 and 257 nm are mainly caused by phenylalanyl, those at 265, 274 and 281 nm by tyrosyl, and those at 291 and 296 nm by tryptophanyl side chains. 2. 2. Only insignificant changes of the tertiary structure seem to occur between pH 6.5 and 8.5. Changes in ellipticity of TMV protein during the pH-induced polymerization reaction suggest that: (1) tyrosyl residues are involved in the binding of subunits, (2) phenylalanyl residues seem to be transferred to a less rigid environment, and (3) tryptophanyl residues are not essential for the reaction. 3. 3. The proteins of several TMV strains and mutants studied have similar far ultraviolet CD spectra and apparently do not differ significantly in their structure. Their near ultraviolet CD spectra are, however, different. Replacements involving aliphatic amino acids do not change considerably the near ultraviolet CD spectra. On the other hand, replacements involving aromatic amino acids have a great effect on the spectra rendering possible identification of CD bands and recognition of the aromatic amino acid residues responsible for optical activity.

An unusual inclusion in plants infected with a tobacco mosaic virus mutant.

The mutant Ni 118 obtained from tobacco mosaic virus (TMV) treated with nitrous acid produces defective coat protein, especially when multiplying in plants kept at 35°. At this temperature, few complete virus particles are formed, and extracts of infected plants contain mostly free infective RNA and insoluble coat protein. At 20°, however, Ni 118 multiplies as well as the type strain of TMV ( Jockusch, 1966 ; Kassanis & Bastow, 1971a ). To gain more information about the conditions in infected cells we examined them by light and electron microscopy. Light microscopy was done by phase-contrast on living cells from epidermal strips of the undersides of Samsun tobacco leaves 1 week after inoculation. For electron microscopy the methods of Milne (1970) were used; pieces of leaf were fixed in glutaraldehyde and then osmium tetroxide, dehydrated in acetone, soaked in uranyl acetate in acetone and embedded in Epon. After sectioning, the material was stained in lead citrate.

Reproduction of temperature-sensitive strains of TMV under restrictive conditions in the presence of temperature-resistant helper strain

Temperature-sensitive (ts) coat protein mutants of tobacco mosaic virus (TMV) Ni 118 and flavum are known to produce insoluble protein and free infective RNA in cells infected at high temperatures. Under conditions of mixed infection these ts-mutants can be complemented by temperature-resistant (tr) strains. Different tr strains (vulgare, A 14, dolichos enation mosaic virus) can serve as helper viruses. It was shown that when Ni 118 was complemented by dolichos enation mosaic virus (DEMV) this effect was due to the masking of Ni 118 genome within DEMV coat protein.

Artificial histidine-containing mutants of tobacco mosaic virus.

Artificial histidine-containing mutants of tobacco mosaic virus.

Recovery of Virulence in a Mutant of Tobacco Mosaic Virus, LN27

A tomato strain of tobacco mosaic virus, TMV-L, was treated with nitrous acid in the extract of infected tobacco leaf, and a mutant was isolated by transferring individual local lesions on Nicotiana glutinosa to tomato seedlings. An isolate, designated as LN27, caused no symptoms on tomato, but when the extract of such symptomless leaves was inoculated on N. glutinosa, and extracts of single-lesions were further inoculated on tobacco plants, a portion of the tobacco plants developed mosaic symptoms after varying periods. Some of the plants which remained symptomless contained virus systemically, while others only in inoculated leaves, or in no parts of the plants. The mosaic symptoms developed on these plants were apparently similar to those caused by the parent virus TMV-L.Lesion productivities of the viruses contained in individual lesions of N. glutinosa were different. The viruses which produced many lesions caused mosaic symptoms on tobacco about 5 days after inoculation. Some of the viruses which produced only a few lesions (2-10 lesions per leaf) caused mosaic symptoms 12-41 days after inoculation, while others caused no symptoms at least during this period.When Xanthi nc tobacco was inoculated with the LN27-infected tomato extract, the leaves of the tobacco showed both distinctly large and small lesions. The viruses from the large lesions caused mosaic symptoms on tomato, while the viruses from the small lesions caused no symptoms for two weeks or more after inoculation, developing symptoms afterwards in a half to a third of them. Inoculation from the tomato which remained symptomless for some time and showed symptoms later gave rise to an increased number of large lesions.It was considered that a part of LN27 somehow recovered its virulence to tobacco and tomato during multiplication.

Serological studies on naturally occurring strains and chemically induced mutants of tobacco mosaic virus.

Abstract The following tobacco mosaic virus strains: type, J14D1, YA, 01, Y-TAMV, G-TAMV, U2, HR, CV4, and 02, as well as sixteen nitrous acid mutants having known primary structural differences from type TMV have been studied by immune-diffusion tests. By means of intragel absorption experiments, antibodies specific for each of several groups of TMV strains were demonstrated. Using protein subunits of different strains, the quaternary structure of the protein was shown to influence the serological properties. A limited number of residue exchanges in the TMV protein coat altered the serological specificity, but only when they occurred at certain critical locations in the peptide chain. A single exchange in a mutant could lead to its differentiation from TMV by the formation of a spur, although in other cases as many as three exchanges left the serological properties unaffected. Serious limitations to the use of qualitative and quantitative serological criteria for purposes of virus classification were found to exist and are discussed.

Die primäre Proteinstruktur Temperatur-sensitiver Mutanten des Tabakmosaikvirus

The protein structure of spontaneous temperature-sensitive mutants of tobacco mosaic virus has been determined. The protein of the mutants was digested with trypsin, the tryptic peptides were isolated and their amino acid composition quantitatively determined. For those tryptic peptides which have amino acid exchanges, the position of the exchange was localized. The experimental results given in the tables are discussed in the following paper.