U1 Strain Research Articles

Expression of the large 5′-proximal cistron of tobacco mosaic virus by 70 S ribosomes during cotranslational disassembly in a prokaryotic cell-free system

In vitro translation of pH 8-washed particles of tobacco mosaic virus (TMV; vulgare, common, or U1 strain) in a cell-free system derived from Escherichia coli (MRE 600) results in a broad size range of TMV RNA-encoded products, including significant amounts of the distinctive 126-kDa polypeptide which is encoded by the first, 5′-proximal open reading frame in TMV RNA and is conventionally expressed by 80 S eukaryotic ribosomes. In an identical prokaryotic translation system, unencapsidated TMV RNA encodes a series of polypeptides of 50 kDa or less. The predominant product of 16–17 kDa frequently appears to comigrate with TMV coat protein. The significance of the former result for mRNA-ribosome interactions and for possible virus-uncoating/early gene expression events within the chloroplasts of infected cells is discussed.

Cotranslational disassembly of a cowpea strain ( Cc) of TMV: Evidence that viral RNA-protein interactions at the assembly origin block ribosome translocation in vitro

Cotranslational disassembly of mixed rod-length populations of tobacco mosaic virus (TMV; vulgare, common or, U1 strain), reveals a reproducible, significant reduction in the level of expression of the intermediate-sized, subgenomic 12-RNA when compared with conventional total viral RNA preparations. I 2-RNA encodes a 30-kDa protein, and recent evidence suggests that I 2-RNA is unusual in that it lacks a 5′-cap structure. In TMV vulgare, the assembly origin is located within the 30-kDa protein-coding region. To resolve which of these structural features might be responsible for the decline in 30-kDa gene expression from packaged 12-RNAs, the products encoded in vitro by packaged or naked genomic and subgenomic RNAs from two strains of TMV, vulgare and a cowpea strain ( Cc or Sunn-hemp mosaic virus), were compared. The results indicate that strong coat protein-RNA interactions, presumed to occur at the assembly origin, dictate the site at which translocation of 80 S ribosomes is inhibited. The implications of this conclusion for virus infection in vivo are discussed.

Comparative study of the U1, U4, and U7 strains of tobacco mosaic virus

Experiments were undertaken to compare three strains of tobacco mosaic virus (TMV) belonging to the tobacco group 1 of the tobamoviruses. Although the U1, U4, and U7 strains of TMV differ significantly in virulence, it was shown that these strains are very closely related. No difference was detected in the major spots of the coat protein tryptic maps and the virions from the three strains shared the same electrophoretic mobility in agarose gels with different buffer systems. The RNA fingerprints of U1 and U7 were indistinguishable and the largest T1 omega fragments of the three strains were identical in size. However, the avirulent U4 RNA fingerprint exhibited a single spot mobility difference from the U1 and U7 RNA fingerprints. This spot corresponded to the T1 phi fragment. This unique fragment is located in the so-called l2 gene between nucleotides 976 and 992 from the 3′ end. This suggests and extends the proposal that the l2 gene is most probably implicated as a factor in the control of virulence (symptomatology).

Polyacrylamide–agarose gel electrophoretic analysis of tobacco mosaic virus disassembly intermediates

The intermediates of disassembly of the U1, U4, U6, and U7 strains of tobacco mosaic virus (TMV) induced by alkali and urea were directly analysed by electrophoresis in composite polyacrylamide (2.0 to 2.5% (w/v)) – agarose (0.5% (w/v)) gels, using a discontinuous buffer system with Tris, borate, EDTA, and sodium chloride (pH 8.3). The results show no difference between the disassembly patterns of the four strains. In the case of alkaline stripping, two new major partially stripped virus (PSV) particles (PSV 5a and PSV 5c) and several minor intermediates were characterized. The overall results indicate that polyacrylamide–agarose gel electrophoresis is superior to agarose gel electrophoresis and to sucrose density gradient ccntrifugation for the analytical separation of PSVs. Polyacrylamide–agarose gel electrophoresis is thus a rather simple, rapid, and efficient method of studying in vitro disassembly of plant viruses.

β-1,3-Glucanase and the spread of tobacco mosaic virus in Nicotiana and Phaseolus

The β-1,3-glucan callose is thought to form a seal which surrounds viral local lesions and prevents viral spread in many plant hosts. Therefore, we investigated the role of host β-1,3-glucanase in facilitating viral spread. The following were compared for lesion size (indicating viral spread rate) and β-1,3-glucanase activity: (i) inoculated excised leaves of Phaseolus vulgaris L. cv. Pinto supplied with sugar (small lesions) or deprived of sugar (large lesions); (ii) Nicotiana glutinosa L. infected with the VM strain (small lesions) or the U1 strain (large lesions) of tobacco mosaic virus; and (iii) Nicotiana sylvestris Spegaz. infected with the VM strain (small lesions) or the U2 strain (large lesions) of tobacco mosaic virus. In all cases, the larger, more rapidly spreading viral lesions did not have significantly higher levels of β-1,3-glucanase activity than the corresponding smaller lesions. Nicotiana sylvestris leaves with a systemic viral infection had a β-1,3-glucanase activity lower than that of leaves with local lesions. Finally, β-1,3-glucanase activity was stimulated to the same extent by a slowly developing abiotic necrosis as by local lesions of tobacco mosaic virus, β-1,3-Glucanase activity may therefore increase during localized viral infection as a result of the wounding associated with necrotic viral lesions.

Cross‐Protection and Multiplication of Mild and Severe Strains of TMV in Tomato Plants

AbstractThe presence of virus having mixed coats has been demonstrated serologically in tomato plants cross‐protected with a mild strain (M 11–16) against the severe (UI) strain of tobacco mosaic virus (TMV). The proportion of virus having mixed coats remained constant throughout at ca. 40% even in top leaves where severe symptoms caused by the challenge virus (U1) first appeared.The symptoms caused by the U1 in doubly‐inoculated plants occurred in the leaves where: the amout of total virus (irrespective of the type of coat) protein was 52% that of U1 strain in singly inoculated plants, the multiplication of the severe U1 strain was reduced to only 9% as compared with the leaves singly inoculated with U1, the ratio of severe to mild strain was increased to near equality although there was a significant reduction in the multiplication of U1in these leaves, the amount of infective U1RNA started to increase.

Study of tobacco mosaic virus in vitro disassembly by sucrose density gradient centrifugation and agarose gel electrophoresis

In vitro disassembly of tobacco mosaic virus (TMV) strains U1, U2, U4, U6, and U7 with alkali and urea was studied by sucrose or sucrose–dimethylsulfoxide (DMSO) density gradient centrifugation and by agarose gel electrophoresis. All strains gave similar decapsidation patterns with both agents when partially stripped virus particles (PSVs) were analyzed by sedimentation and electrophoresis. However, U6 was more sensitive to decapsidation than the other strains and U2 exhibited resistance to decapsidation. Agarose gel electrophoresis of TMV decapsidation products allowed the detection of several classes of PSVs in addition to aggregation products involving PSVs and monomer particles. Agarose gel electrophoresis is thus very rapid and useful for analysis of TMV disassembly products especially when aggregation phenomena and kinetic studies with numerous samples are considered.

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.

Strains and mutants of tobacco mosaic virus are both found in virus derived from single-lesion-passaged inoculum

An analysis has been made of variants found in preparations of the common (U1) strain of tobacco mosaic virus (TMV) which had been obtained after the inoculum had been passaged through a necrotic local lesion. These lesions were induced with a minimal number of virions (3000 or 30,000) per inoculation point. The relationships of the variants to the U1 strain were analyzed both by nucleic acid hybridization using complementary DNA in nuclease S1 assays and by two-dimensional RNA fingerprinting. Two classes of variants were obtained: Members of one class, because of their close sequence homology to the U1 strain, were considered to arise by mutation of the U1 strain. These variants were isolated from chlorotic or necrotic spots which developed on the leaves of U1-infected plants. The other class represented distantly related strains of TMV which are presumed to have been passaged as contaminants along with the U1 strain, even though the virus from which the inoculum was obtained was itself generated via a single local lesion from minimum dose inoculation. Such data thus cast suspicion on the fidelity of local lesion passage as a means of getting biologically “pure” virus, viz (1) contaminants could be passaged along with the major virus (normally they would not be seen, but they might be expressed under the appropriate environmental conditions), and (2) the high rate of transcriptional infidelity known to occur during RNA synthesis results in a heterogeneous viral RNA population but with one predominant genotype. Occasionally, mutants with a new phenotype may be expressed (as chlorotic or necrotic spots on a leaf of mosaic symptom plants, for example).

The H protein isolated from tobacco mosaic virus reassociates with virions reconstituted in vitro

Virions of two strains of tobacco mosaic virus (U1 and Cc) have associated with them a small amount of a minor protein called H protein (A. Asselin and M. Zaitlin, 1978, Virology 91, 173-181), now known to be related to the viral coat protein (C.W. Collmer, V.M. Vogt, and M. Zaitlin, 1983, Virology 126, 429-448.). In the present study, a quantification technique involving disruption of virions followed by direct analysis of the component parts on SDS polyacrylamide gels was used to confirm an average of one molecule of H protein per virion for U1 TMV. H protein was separated from coat protein and purified by electrofocusing in a flatbed of granulated gel under stringent dissociating conditions. When assayed in the presence of urea, H protein has a pI of approximately 5.4, coat protein has a pI of approximately 4.9. Proteinase K-treated TMV RNA and H-protein-free TMV coat protein were reconstituted in vitro with or without H protein and the resulting virions were analyzed. A small amount of H protein reassociated with virions reconstituted in vitro (less than 10% of the amount found in native virions) and became resistant to degradation by trypsin, but such virions were no different from virions reconstituted without H protein in terms of yield of reconstituted particles or infectivity. In mixed reconstitution experiments with RNA and coat protein from strains U1 and Cc in all four possible combinations and with U1 H protein, the H protein always associated with the U1 coat protein. This demonstrated U1-H protein affinity for a specific coat protein rather than a specific RNA. It is unlikely that H protein functions in the early stages of viral infection, although the possibility of its having some other role in the life cycle of TMV remains.

H protein, a minor protein of TMV virions, containssequences of the viral coat protein

H protein, a minor protein found associated with virions of tobacco mosaic virus (TMV) at an average of about one copy per virion and previously believed to be host-coded (Asselin and Zaitlin, 1978, Virology 91, 173-181), has been shown to contain sequences of the viral capsid protein. Two-dimensional tryptic peptide maps of 125I-labeled H protein (Mr 26,500) and coat protein (Mr 17,500) from TMV strains U1 and Dahlemense show that the respective H proteins contain most if not all of the labeled peptides of the coat proteins in addition to 2-3 unique peptides. The H proteins also contain unique antigenic determinants, as antibodies can be isolated which react strongly with the H protein but not with the coat protein of Dahlemense TMV. Finally, amino acid composition analysis of the U1-TMV H protein has shown the presence of methionine and histidine, amino acids not present in the coat protein of that strain. H protein appears to contain the same NH2 terminus as coat protein, as there is an H protein tryptic peptide that both comigrates in a two-dimensional system and produces the same acid cleavage product as the NH2-terminal tryptic peptide of coat protein. H protein also seems to have the same COOH terminus as coat protein, as cyanogen bromide digestion of Dahlemense-TMV coat protein and H protein indicates that each has a methionine about 12 amino acids from one terminus (known to be the COOH terminus of the coat protein). Thus, H protein is not structurally equivalent to coat protein with an addition on either its NH2 or COOH terminus. However, H protein does not appear to be a noncovalent aggregate of coat protein and some other protein. Rather, the model we favor for H protein structure is that of a branched fusion product between coat protein and another polypeptide of host or viral origin.

Growth of Pseudomonas aeruginosa mutants lacking glutamate synthase activity.

Mutant strains SU1, SU4, and US1 lacking glutamate synthase (GOGAT) activity were isolated from strains of P. aeruginosa for which histidine is a growth rate-limiting source of nitrogen. Strains SU1 and SU4 were unable to grow when a low concentration of ammonia and a variety of compounds, including histidine, were supplied as sole sources of nitrogen. A revertant of strain SU1, strain 39, produced no GOGAT but high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase and had restored ability to grow on a limited number of nitrogen sources. Strain US1 grew at the same rate in histidine medium as did its parent; it was derepressed for glutamine synthase synthesis, and histidase was less sensitive to repression by ammonia than in the parent strain. We conclude that GOGAT is not essential for growth on histidine but high levels of glutamine synthase are required nd high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase can sustain growth at low concentrations of ammonia in the absence of GOGAT.

Binding of ribosomes to linear and circular forms of the 5'-terminal leader fragment of tobacco-mosaic-virus RNA.

The sequence of the 5'-terminal leader fragment preceding the AUG codon in the RNA of tobacco mosaic virus (TMV), tomato strain, SPS isolate, has been determined. This RNA, similarly to the RNAs of the U1 and Dahlemense strains of TMV [Kukla et al. (1979) Eur. J. Biochem. 98, 61--66] has the 7-methylguanosine(5')triphospho(5')guanosine cap separated from the initiation codon by a long stretch of nucleotides devoid of guanosine residues. The RNase-T1-resistant 73-nucleotide-long leader fragment of TMV RNA from the SPS isolate was assayed for its ability to interact with eukaryotic and prokaryotic ribosomes. The linear fragment, labelled either at its 5' or 3' end, efficiently formed disome initiation complexes when incubated with wheat-germ protein-synthesis extract. In contrast to its linear counterpart, the circular covalently closed RNA leader fragment, obtained in a reaction catalysed by T4 RNA ligase, was unable to interact with wheat germ ribosomes. Both kinds of leader fragment bound equally well to Escherichia coli 70-S ribosomes. The results offer further support to the notion that in eukaryotic initiation the free 5' end (either capped or uncapped) is required for mRNA interaction with ribosomes. Furthermore, they suggest that both ribosomes found in disome initiation complexes with the TMV RNA leader fragment enter the mRNA sequentially via the free 5' terminus.

Sequence and Antigenic Activity of the Region 93 to 113 of the Coat Protein of Strain U2 of Tobacco Mosaic Virus

SUMMARY The amino acid sequence of the coat protein of the U2 strain of tobacco mosaic virus (TMV) has been re-examined and completed. Four incorrect residue allocations in the published U2 sequence were identified. These were located in the region corresponding to residues 96 to 105. In addition, the identity of residues 106 to 112 was also determined. This latter region is of particular interest in antigenic studies, since the homologous region 108 to 112 in TMV (common strain) corresponds to an antigenic determinant of the depolymerized coat protein. Tryptic peptide 6 of strain U2 (residues 93 to 113) also contains an antigenic determinant, as shown by its ability to inhibit the reaction between U2 protein and specific antibodies.

Titration behaviour of three strains of tobacco mosaic virus

Hydrogen ion titration curves of the virions and proteins of three strains of tobacco mosaic virus (Y-TAMV, U2, and cowpea) were measured in the absence and the presence of Ca 2+, Mg 2+, or Mn 2+ ions, and compared with the analogous curves for the type strain (vulgare). Extinction coefficients were also measured for all four strains' virions and proteins. Y-TAMV is very like vulgare in its cation affinities: the virion has probably three groups per protein subunit that titrate near neutral pH and significantly bind metal ions; the RNA-free protein has very little affinity for Ca 2+, although moderate Ca 2+ concentrations favour the existence of larger polymers. U2 and cowpea strain virions bind cations significantly more strongly than do Y-TAMV or vulgare virions: their polymerized proteins, too, have significant affinities for Ca 2+ ions, which make their titration and sedimentation behaviours relatively sensitive to added calcium. These cation-binding differences correspond well with the differences between the strains' protein sequences. The features common to all four strains are that the virions are apparently structurally invariant and have at least one site per subunit with Ca 2+ affinity in the region of 10 −5 M, while the RNA-free proteins lack the high-affinity sites but have weaker Ca 2+ affinities in the region of 10 −3 M. Some of the cation-binding sites probably lie near the central holes of the virions.

The role of the fibrillar component of the surface sheath in the morphogenesis of Dictyostelium discoideum

We have isolated a fibrillar component of the surface sheath of Dictyostelium discoideum by virtue of its insolubility in 9 M urea-2% sodium dodecyl sulfate (US). The US-insoluble material is primarily composed of cellulose, but also contains other carbohydrate components, protein, and lipid. Evidence is presented that the US-insoluble material is a component of the sheath. Sheath isolated from mutant strains lacking the developmentally regulated N-acetylglucosaminidase (NAG), α-mannosidase, or β-glucosidase activities is similar in composition to sheath isolated from wildtype strain. Strains lacking NAG are unable to migrate normally. This may result from the markedly lower crystallinity of the cellulose in the US-insoluble sheath isolated from these mutants. Strains lacking α-mannosidase or β-glucosidase migrate normally and the crystallinity of the sheath cellulose is not significantly below that of the wildtype. The correlation between the lower crystallinity of cellulose and the inability of strains lacking NAG to migrate suggests that crystallinity is physiologically important and that the degree of crystallinity is controlled by an enzymatic, mutable process. Strains U1 and UN1, which have <1% of the wildtype activity of uridine diphosphate glucose pyrophosphorylase activity, develop in a morphologically normal fashion to the slug stage. However, they cannot form stalk or spore cells, nor can they produce cellulose. These strains do not produce any detectable US-insoluble sheath and are fragile and unable to migrate. A continuous, nonfibrillar sheath surrounds the aggregates and is sufficient for normal morphogenesis up to the slug stage. The fibrillar component gives the aggregate the added rigidity required during migration.

A study of nucleotide sequence homology between strains of tobacco mosaic virus

Nucleotide sequence similarities between the genomes of strains of tobacco mosaic virus have been assessed by a competition-hybridization test. It was found that strains fall into several groups with nucleotide sequences being indistinguishable within a group and without similarity between groups. The strains fall into the same groups whether these are based on capsid protein properties or on nucleotide sequence homology. The two groups represented by strains U1 and dahlemense have capsid proteins which are the most alike of any of the intergroup comparisons, having differences in only 29 or 18% of the 158 sequential amino acid positions and, yet, their nucleic acids show no homology in the competition-hybridization test. Rabbit and mouse globins differ to about the same extent as U1 and dahlemense capsid protein, but in this case, considerable homology has been detected between their genes (Gummerson, R. S., and Williamson, R., Nature (London) 297, 265–267, 1974). Possible reasons for the difference in the two systems are discussed.

Mixed infection with two tobamoviruses: the formation of particles containing the coat protein messenger RNAs of either virus.

Plants mixedly infected with the U2 strain of tobacco mosaic virus (T2MV) and sunnhemp mosaic virus (SHMV) and grown at 35 degrees, yield particles of the same modal lengths (300 and 40 nm) as those found in plants singly infected with SHMV, but not in plants infected with T2MV, which yield only the long particles. At least some of the particles produced in mixedly infected plants contain coat proteins of both viruses. When RNAs from these particles are translated in vitro the coat proteins of both viruses are produced, although when a mixture of RNAs from particles of SHMV and T2MV, grown separately, are translated in vitro only SHMV protein is produced. These and other results suggest that the short particles produced in mixedly infected plants contain both coat protein messengers.

Preparation of an isomorphous heavy-atom derivative of tobacco mosaic virus by chemical modification with 4-sulpho-phenylisothiocyanate

The reaction of the vulgare and U2 strains of tobacco mosaic virus with 4-sulpho-phenylisothiocyanate has been investigated. The coat protein of the U2 strain has a proline residue at its N-terminus and a lysine residue at position 53. Whereas both residues could be reacted with 4-sulpho-phenylisothiocyanate in the isolated coat protein, only proline-1 was modified during treatment of the intact virus with the same reagent, thereby showing that the loss of reactivity of the ϵ-amino group of lysine-53 is a consequence of the virus structure. The 4-sulpho-phenylthiocarbamoyl derivative of amino groups shows considerable tautomerism and, as a consequence, it proved possible to prepare a heavy-atom derivative of the intact U2 strain in which methyl mercury nitrate was bound by the modified N-terminal residue of the coat protein. On the other hand, when the intact vulgare strain was treated with 4-sulphophenylisothiocyanate, little or no modification of the ϵ-amino groups of the two lysine residues (positions 53 and 68) per polypeptide chain was observed. Taking into account previous studies on the reactivity of the amino groups of the coat protein in tobacco mosaic virus vulgare and assuming that all strains and mutants have closely similar three-dimensional structures, these experiments suggest that the N-terminal residue is more exposed (i.e. probably nearer the virus “surface”) than the side-chain of lysine-68, which in turn is more accessible than the side-chain of lysine-53. This interpretation is readily compatible with the results of X-ray diffraction analysis carried out on these chemically modified viruses (Mandelkow & Holmes, 1974) and lends support to the hope that such methods of preparing heavy-atom derivatives of proteins will be of general use.

Pseudovirions of tobacco mosaic virus

Tobacco mosaic virus (TMV) preparations contain a small percentage of pseudovirions. These particles, which are thought to consist of host RNA encapsidated with TMV protein, can be separated from virus particles by electrophoresis in 0.5% agarose gel. Pseudovirion RNA is smaller than and has a broader size distribution than viral RNA. It is complementary to host DNA, more so to chloroplast DNA than to nuclear DNA. Strains of TMV have different pseudovirion contents, with the U2 strain having the greatest proportion (2–2.5%) of the strains tested. U2 pseudovirion RNA is complementary to about 20% of tobacco chloroplast DNA and 0.4% of tobacco nuclear DNA. A large proportion of pseudovirion RNA is complementary to both chloroplast and nuclear DNA, suggesting that chloroplast DNA has many nucleotide sequences in common with nuclear DNA.