Properties of the coat protein of the tobacco mosaic virus Kazakh isolate K1

A study was made of the coat protein (CP) of thermosensitive semidefective tobacco mosaic virus strain K1 (TMV-K1). In contrast to CP of other TMV strains, K1 CP showed high nonspecific aggregation and did not form normal two-layered cylindrical aggregates. In none of the conditions tested, K1 CP formed virions with cognate K1 RNAin vitro. The abnormal properties were attributed to substitution Lys53→Glu differentiating the K1 CP from those of other tobamoviruses. It is assumed that the high structural plasticity allows the tobamovirus virions to incorporate CP subunits even with unfavorable amino acid changes.     

The antigenicity of tobacco mosaic virus

The antigenic properties of the tobacco mosaic virus (TMV) have been studied extensively for more than 50 years. Distinct antigenic determinants called neotopes and cryptotopes have been identified at the surface of intact virions and dissociated coat protein subunits, respectively, indicating that the quaternary structure of the virus influences the antigenic properties. A correlation has been found to exist between the location of seven to ten residue-long continuous epitopes in the TMV coat protein and the degree of segmental mobility along the polypeptide chain. Immunoelectron microscopy, using antibodies specific for the bottom surface of the protein subunit, showed that these antibodies reacted with both ends of the stacked-disk aggregates of viral protein. This finding indicates that the stacked disks are bipolar and cannot be converted directly into helical viral rods as has been previously assumed. TMV epitopes have been mapped at the surface of coat protein subunits using biosensor technology. The ability of certain monoclonal antibodies to block the cotranslational disassembly of virions during the infection process was found to be linked to the precise location of their complementary epitopes and not to their binding affinity. Such blocking antibodies, which act by sterically preventing the interaction between virions and ribosomes may, when expressed in plants, be useful for controlling virus infection.     

Modification of Tobacco Mosaic Virus by Polyornithine and Lecithin

Combined action of polyornithine and lecithin modified tobacco mosaic virus (TMV) virions making them sensitive to ribonuclease (RNase), pronase or Triton X-100. Sedimentational analysis and examination of the fluorescence spectrum revealed that the reaction product obtained after RNase treatment of modified TMV was a three-component complex made of coat protein, polyornithine and lecithin. The minimum requirement for the modification was completely fulfilled by cetyltrimethylammonium bromide, suggesting that a positively charged nitrogen group and an alkyl group of moderate size, C_10–18, are necessary components. These components react with the surface region of TMV which is considered to have an important role in connecting coat protein subunits in TMV virions.     

Conformational changes preceding decapsidation of bromegrass mosaic virus under hydrostatic pressure: a small-angle neutron scattering study

The stability of bromegrass mosaic virus (BMV) and empty shells reassembled in vitro from purified BMV coat protein was investigated under hydrostatic pressure, using solution small-angle neutron scattering. This technique allowed us to monitor directly the dissociation of the particles, and to detect conformational changes preceding dissociation. Significant dissociation rates were observed only if virions swelled upon increase of pressure, and pressure effects became irreversible at very high-pressure in such conditions. At pH 5.0, in buffers containing 0.5 M NaCl and 5 mM MgCl(2), BMV remained compact (radius 12.9 nm), dissociation was limited to approximately 10 % at 200 MPa, and pressure effects were totally reversible. At pH 5.9, BMV particles were slightly swollen under normal pressure and swelling increased with pressure. The dissociation was reversible to 90 % for pressures up to 160 MPa, where its rate reached 28 %, but became totally irreversible at 200 MPa. Pressure-induced swelling and dissociation increased further at pH 7.3, but were essentially irreversible. The presence of (2)H(2)O in the buffer strongly stabilized BMV against pressure effects at pH 5.9, but not at pH 7.3. Furthermore, the reversible changes of the scattered intensity observed at pH 5.0 and 5.9 provide evidence that pressure could induce the release of coat protein subunits, or small aggregates of these subunits from the virions, and that the dissociated components reassociated again upon return to low pressure. Empty shells were stable at pH 5.0, at pressures up to 260 MPa. They became ill-shaped at high-pressure, however, and precipitated slowly after return to normal conditions, providing the first example of a pressure-induced conformational drift in an assembled system.     

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 0.2M acetic acid, pH 4.6, and was passed through membrane filter Nanosep 300K 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.     

Optical rotatory dispersion by 5 viruses of the tobacco mosaic virus group and their components

Optical rotatory dispersion (ORD) spectra in 250 to 350 nm region were measured for preparations of five TMV-like viruses (TMV vulgare, HR and U2 strains of TMV dolihosenation mosaic virus and cucumber virus 4) and also for RNA and protein preparations of these viruses. The data obtained testify against the possibility that the double peak with maxima at 286 and 293 nm observed in ORD of all the five viruses is due to interaction of tryptophan residues in virus coat protein with the RNA of the virul particle. The spectra of intravirus RNA of the five viruses, calculated as the difference between ORD of the intact virus and of its coat protein, were found to differ significantly from each other and from ORD of free RNA. ORD spectra of hybrid viruses, reconstituted from RNA of one virus and coat protein of another, proved to be identical to the ORD of the virus, whose protein was used in reconstitution. We suppose that the difference in ORD of the intravirus RNA of the five viruses reflect differences of RNA-protein interactions in them.     

Proteomic analysis of tobacco mosaic virus-infected tomato (Lycopersicon esculentum M.) fruits and detection of viral coat protein

Tobacco mosaic virus (TMV) is a widespread plant virus from the genus Tobamovirus that affects tobacco and tomato plants causing a pathology characterised by cell breakage and disorganisation in plant leaves and fruits. In this study we undertook a proteomic approach to investigate the molecular and biochemical mechanisms potentially involved in tomato fruit defence against the viral infection. The comparison of 2-D gels from control and TMV-infected but asymptomatic tomato fruits revealed changes in several proteins. The differential expression of peptidases, endoglucanase, chitinase and proteins participating in the ascorbate-glutathione cycle in infected fruits suggests that pathogenesis-related proteins and antioxidant enzymes may play a role in the protection against TMV infection. TMV coat protein appeared as a prominent spot in 2-D gels from TMV-infected asymptomatic fruits. A Triton X-114 phase-partitioning step of tomato protein extracts favoured the solubilisation of TMV coat protein and the enrichment of two aminopeptidases not present in control fruits. PMF and MS/MS data of the 2-D gel-isolated TMV coat protein is proposed as a powerful analysis method for the simultaneous tobamovirus detection, species determination and strain differentiation in virus-infected fruit commodities.     

Comparative Study on Coat Proteins of an Attenuated, a Temperature Sensitive and Their Parent Strains of Tobacco Mosaic Virus

An attenuated strain (L₁₁A) of tobacco mosaic virus (TMV) induces no remarkable symptoms on tomato plants (Goto and Nemoto 1971) and has been used to protect tomato against virulent strains of TMV (Oshima 1981), A temperature sensitive strain (Ls1) of TMV was isolated and found to have a malfunction of virus movement from cell to cell (NISHI-GUCHI et al. 1978, 1980). Those two strains are derived from a wild virulent strain (L). Coat proteins of them were compared with one another and with that of Dahlemense (D) strain of TMV, in order to see whether coat protein was associated with their respective characters. The coat proteins of the four strains behaved similar in both SDS-polyacrylamide gel and 8 M urea polyacrylamide gel electrophoresis, suggesting that they are similar in molecular weight and charging effect in the gels. There was no significant difference in chromatographic pattern of tryptic peptides among the four strains. Amino acid compositions of tryptic peptides revealed that three strains, L₁₁A, Ls1 and L, were identical to one another and that they differed from D slightly. These results suggest that coat protein is related neither to virus attenuation of L₁₁A nor to the malfunction of Ls1.     

The 2b silencing suppressor of a mild strain of Cucumber mosaic virus alone is sufficient for synergistic interaction with Tobacco mosaic virus and induction of severe leaf malformation in 2b-transgenic tobacco plants

Tobacco plants infected simultaneously by Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) are known to produce a specific synergistic disease in which the emerging leaves are filiformic. Similar developmental malformations are also caused to a lesser extent by the severe strains (e.g., Fny) of CMV alone, but mild strains (e.g., Kin) cause them only in mixed infection with TMV. We show here that transgenic tobacco plants expressing 2b protein of CMV-Kin produce filiformic symptoms when infected with TMV, indicating that only 2b protein is needed from CMV-Kin for this synergistic relationship. On the other hand, transgenic plants that express either the wild-type TMV genome or a modified TMV genome with its coat protein deleted or movement protein (MP) inactivated also develop filiformic or at least distinctly narrow leaves, while plants expressing the MP alone do not develop any malformations when infected with CMV-Kin. These results show that either TMV helicase/replicase protein or active TMV replication are required for this synergistic effect. The effect appears to be related to an efficient depletion of silencing machinery, caused jointly by both viral silencing suppressors, i.e., CMV 2b protein and the TMV 126-kDa replicase subunit.     

Single-Molecule Force Spectroscopy Study on the Mechanism of RNA Disassembly in Tobacco Mosaic Virus

To explore the disassembly mechanism of tobacco mosaic virus (TMV), a model system for virus study, during infection, we have used single-molecule force spectroscopy to mimic and follow the process of RNA disassembly from the protein coat of TMV by the replisome (molecular motor) in vivo, under different pH and Ca²⁺ concentrations. Dynamic force spectroscopy revealed the unbinding free-energy landscapes as that at pH 4.7 the disassembly process is dominated by one free-energy barrier, whereas at pH 7.0 the process is dominated by one barrier and that there exists a second barrier. The additional free-energy barrier at longer distance has been attributed to the hindrance of disordered loops within the inner channel of TMV, and the biological function of those protein loops was discussed. The combination of pH increase and Ca²⁺ concentration drop could weaken RNA-protein interactions so much that the molecular motor replisome would be able to pull and disassemble the rest of the genetic RNA from the protein coat in vivo. All these facts provide supporting evidence at the single-molecule level, to our knowledge for the first time, for the cotranslational disassembly mechanism during TMV infection under physiological conditions.     

Polymorphism of turnip yellow mosaic virus empty shells and evidence for conformational changes occurring after release of the viral RNA. A differential scanning calorimetric study.

Turnip yellow mosaic virus (TYMV) is a small isometric plant virus which decapsidates by releasing its RNA through a hole in the capsid, leaving behind an empty shell [R. E. F. Matthews and J. Witz, (1985) Virology 144, 318-327]. Similar empty shells (artificial top component, ATC) can be obtained by submitting the virions to various treatments in vitro. We have used differential scanning calorimetry, analytical sedimentation, and electron microscopy to investigate the thermodenaturation of natural empty shells (NTC, natural top component) present in purified virus suspensions, and of several types of ATCs. ATCs divided in two major classes. Those obtained by alkaline titration, by the action of urea or butanol behaved as NTC: their thermograms contained only one peak corresponding to the irreversible dissociation of the shells and the denaturation of the coat protein. The temperature of this unique transition varied significantly with pH, from 71 degrees C at pH 4.5 to 84 degrees C at pH 8.5. The thermograms of ATCs obtained by freezing and thawing, or by the action of high pressure, contained two peaks: shells dissociated first into smaller protein aggregates at 57 degrees C (at pH 5.0) to 61 degrees C (at pH 8.5), which denatured at the temperature of the unique transition of NTC. Shells obtained by heating virions to 55 degrees C at pH 7.6, changed conformation after the release of the viral RNA, as upon continuous heating to 95 degrees C, their thermograms were similar to those of the shells obtained by freezing and thawing, whereas after purification they behaved like NTC. Structural implications of these observations are discussed.     

β-structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation

Conversion of the rod-like tobacco mosaic virus (TMV) virions into “ball-like particles” by thermal denaturation at 90–98?°C had been described by R.G. Hart in 1956. We have reported recently that spherical particles (SPs) generated by thermal denaturation of TMV at 94–98?°C were highly stable, RNA-free, and water-insoluble. The SPs were uniform in shape but varied widely in size (53–800?nm), which depended on the virus concentration. Here, we describe some structural characteristics of SPs using circular dichroism, fluorescence spectroscopy, and Raman spectroscopy. It was found that the structure of SPs protein differs strongly from that of the native TMV and is characterized by coat protein subunits transition from mainly (about 50%) α-helical structure to a structure with low content of α-helices and a significant fraction of β-sheets. The SPs demonstrate strong reaction with thioflavin T suggesting the formation of amyloid-like structures.     

Single-Molecule Force Spectroscopy Study on the Mechanism of RNA Disassembly in Tobacco Mosaic Virus

To explore the disassembly mechanism of tobacco mosaic virus (TMV), a model system for virus study, during infection, we have used single-molecule force spectroscopy to mimic and follow the process of RNA disassembly from the protein coat of TMV by the replisome (molecular motor) in vivo, under different pH and Ca²⁺ concentrations. Dynamic force spectroscopy revealed the unbinding free-energy landscapes as that at pH 4.7 the disassembly process is dominated by one free-energy barrier, whereas at pH 7.0 the process is dominated by one barrier and that there exists a second barrier. The additional free-energy barrier at longer distance has been attributed to the hindrance of disordered loops within the inner channel of TMV, and the biological function of those protein loops was discussed. The combination of pH increase and Ca²⁺ concentration drop could weaken RNA-protein interactions so much that the molecular motor replisome would be able to pull and disassemble the rest of the genetic RNA from the protein coat in vivo. All these facts provide supporting evidence at the single-molecule level, to our knowledge for the first time, for the cotranslational disassembly mechanism during TMV infection under physiological conditions.     

Coordinated two-disk nucleation, growth and properties, of virus-like particles assembled from tobacco-mosaic-virus capsid protein with poly(A) or oligo(A) of different length

Assembly of nucleoprotein rods from tobacco mosaic virus (TMV) coat protein and poly(A) depends on the presence of 20S disks in a manner very similar to nucleation and growth of virions in reconstitution with TMV RNA. Products assembled with (A) approximately equal to 5000 appear to have the same buoyant density in CsCl, the same nucleotide/protein ratio and the same nuclease stability, as reconstituted and native TMV. Their rate of formation is very similar to the rate of reconstitution with TMV RNA when high-molecular-mass (A) approximately equal to 5000 is used, but becomes a function of chain length particularly with (A) less than or equal to 185. The composition of assembly products can be described sufficiently with the relation between number of capsid polypeptide monomers/particle, np, to the number of nucleotide residues/chain, nnt, of np = 1/3 (nnt + 50) with two important restrictions: (1) particles of less than four turns of helically arranged capsid subunits are unstable, and (2) particles with about 150 or less nucleotides per chain deviate in structure from mature virus and virus-like (= longer) assembly products. This is indicated by changes in both buoyant density in CsCl and optical properties, while 'dislocation' of the disk to the helical arrangement of capsid subunits ('helicalization') and nuclease stability already become established with chains as short as (A) approximately equal to 58 +/- 20. Consequently, we suggest that assembly proceeds through three distinct phases: (1) nucleation (resulting in helicalization) by interaction of nucleic acid with the first disk; (2) stabilization of the primary (unstable!) nucleation complex by addition of a second disk and formation of a four-turn virus-like and stable nucleoprotein helix, which is then fit for (3) elongation by addition of further disks. The question of what makes the TMV protein disk select specifically TMV RNA during virion assembly is discussed in some detail.     

Historical overview of research on the tobacco mosaic virus genome: genome organization, infectivity and gene manipulation

Early in the development of molecular biology, TMV RNA was widely used as a mRNA [corrected] that could be purified easily, and it contributed much to research on protein synthesis. Also, in the early stages of elucidation of the genetic code, artificially produced TMV mutants were widely used and provided the first proof that the genetic code was non-overlapping. In 1982, Goelet et al. determined the complete TMV RNA base sequence of 6395 nucleotides. The four genes (130K, 180K, 30K and coat protein) could then be mapped at precise locations in the TMV genome. Furthermore it had become clear, a little earlier, that genes located internally in the genome were expressed via subgenomic mRNAs. The initiation site for assembly of TMV particles was also determined. However, although TMV contributed so much at the beginning of the development of molecular biology, its influence was replaced by that of Escherichia coli and its phages in the next phase. As recombinant DNA technology developed in the 1980s, RNA virus research became more detached from the frontier of molecular biology. To recover from this setback, a gene-manipulation system was needed for RNA viruses. In 1986, two such systems were developed for TMV, using full-length cDNA clones, by Dawson's group and by Okada's group. Thus, reverse genetics could be used to elucidate the basic functions of all proteins encoded by the TMV genome. Identification of the function of the 30K protein was especially important because it was the first evidence that a plant virus possesses a cell-to-cell movement function. Many other plant viruses have since been found to encode comparable 'movement proteins'. TMV thus became the first plant virus for which structures and functions were known for all its genes. At the birth of molecular plant pathology, TMV became a leader again. TMV has also played pioneering roles in many other fields. TMV was the first virus for which the amino acid sequence of the coat protein was determined and first virus for which cotranslational disassembly was demonstrated both in vivo and in vitro. It was the first virus for which activation of a resistance gene in a host plant was related to the molecular specificity of a product of a viral gene. Also, in the field of plant biotechnology, TMV vectors are among the most promising. Thus, for the 100 years since Beijerinck's work, TMV research has consistently played a leading role in opening up new areas of study, not only in plant pathology, but also in virology, biochemistry, molecular biology, RNA genetics and biotechnology.     

Flow dichroic spectra of tobacco mosaic virus and their protein assemblies

Ultraviolet flow dichroisms of tobacco mosaic virus (TMV), TMV-RNA and TMV-protein were measured using three strains of TMV. 1. Large positive dichroisms were observed with three strains of TMV, namely ordinary, bean form of bean, and tomato strains (TMV-OM, TMV-B and TMV-T, respectively) at about 255, 276, 284 and 290 nm. The positive dichroisms were confirmed with reconstituted protein assemblies of TMV-OM and TMV-B at about 276, 284 and 290 nm where tyrosine and tryptophan residues of these proteins contribute. These results show that the electronic transition moments of their base groups and aromatic groups are nearly parallel to the polymer axis. It is suggested that there is a strong interaction between the base groups of RNA and aromatic groups of amino acid residues in TMV. 2. A small negative dichroism was observed near 296-300 nm with intact TMV-OM and TMV-T and with the reconstituted protein polymer of TMV-OM. But negative dichroism was not observed either with the intact virus or the protein assembly of TMV-B. 3. Isolated RNA from TMV-OM, TMV-B and TMV-T showed no dichroism. The configuration of RNA in TMV appears to be imposed on it by its packing with the protein.     

Tritium planigraphy comparative structural study of tobacco mosaic virus and its mutant with altered host specificity.

Spatial organization of wild-type (strain U1) tobacco mosaic virus (TMV) and of the temperature-sensitive TMV ts21-66 mutant was compared by tritium planigraphy. The ts21-66 mutant contains two substitutions in the coat protein (Ile21-->Thr and Asp66-->Gly) and, in contrast with U1, induces a hypersensitive response (formation of necroses) on the leaves of plants bearing a host resistance gene N' (for example Nicotiana sylvestris); TMV U1 induces systemic infection (mosaic) on the leaves of such plants. Tritium distribution along the coat protein (CP) polypeptide chain was determined after labelling of both isolated CP preparations and intact virions. In the case of the isolated low-order (3-4S) CP aggregates no reliable differences in tritium distribution between U1 and ts21-66 were found. But in labelling of the intact virions a significant difference between the wild-type and mutant CPs was observed: the N-terminal region of ts21-66 CP incorporated half the amount of tritium than the corresponding region of U1 CP. This means that in U1 virions the CP N-terminal segment is more exposed on the virion surface than in ts21-66 virions. The possibility of direct participation of the N-terminal tail of U1 CP subunits in the process of the N' hypersensitive response suppression is discussed.     

The 5′ Cap of Tobacco Mosaic Virus (TMV) Is Required for Virion Attachment to the Actin/Endoplasmic Reticulum Network During Early Infection

Almost nothing is known of the earliest stages of plant virus infections. To address this, we microinjected Cy3 (UTP)‐labelled tobacco mosaic virus (TMV) into living tobacco trichome cells. The Cy3‐virions were infectious, and the viral genome trafficked from cell to cell. However, neither the fluorescent vRNA pool nor the co‐injected green fluorescent protein (GFP) left the injected trichome, indicating that the synthesis of (unlabelled) progeny viral (v)RNA is required to initiate cell‐to‐cell movement, and that virus movement is not accompanied by passive plasmodesmatal gating. Cy3‐vRNA formed granules that became anchored to the motile cortical actin/endoplasmic reticulum (ER) network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actin‐dependent RNA movement. The 5′ methylguanosine cap was shown to be required for vRNA anchoring to the actin/ER. TMV vRNA lacking the 5′ cap failed to form granules and was degraded in the cytoplasm. Removal of the 3′ untranslated region or replicase both inhibited replication but did not prevent granule formation and movement. Dual‐labelled TMV virions in which the vRNA and the coat protein were highlighted with different fluorophores showed that both fluorescent signals were initially located on the same ER‐bound granules, indicating that TMV virions may become attached to the ER prior to uncoating of the viral genome.     

Die prim?re Proteinstruktur von St?mmen des Tabakmosaikvirus

Summary In contrast to chemically induced mutants of tobacco mosaic virus (TMV) in which we have found replacement of one or at most of two amino acids per coat protein chain, the protein chains of naturally occurring TMV strains differ from each other in numerous positions. The complete amino acid sequence of the naturally occurring TMV straindahlemense isolated byMelchers (1940) has been determined. It differs in 30 of the 158 amino acid positions from the TMV wild strainvulgare (Fig. 1). This is the first case in which complete amino acid sequences of the coat proteins of two virus strains can be compared. Such a comparison permits conclusions about the structure of the protein subunits and about certain aspects of the genetic code to be drawn.The electrophoretic mobility curves for the virus rods and the A proteins ofvulgare anddahlemense (Fig. 4) can be explained on the basis of the amino acid sequences of the two strains. Spatial distribution of the positive and negative groups within the protein subunits are discussed. One particular segment of the protein chain appears to be so important for the secondary and/or tertiary structure of the protein subunit that amino acid replacements within this segment in general lead to a loss of infectivity.The 46 cases in which we have exactly located the positions of amino acid differences betweenvulgare and various TMV mutants and strains are summarized in Table 1. Combination of the data in Table 1 with the base compositions of the triplets as obtained from the cell free system ofE. coli permits conclusions about the nucleotide sequence within the triplets to be drawn. The triplets shown in Table 2 represent, at present, the best agreement between the data from the cell free system and the work with TMV mutants.

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Early Events in the Infection of Permissive Cells with Simian Virus 40: Adsorption, Penetration, and Uncoating

The early events in the interaction of simian virus 40 (SV40) with permissive cells were investigated. Evidence is presented that 30 min after infection intact virions penetrate the nuclei of infected cells. The uncoating of the virus is carried out in the nuclei with a complete dissociation of the viral genome from the protein coat. Opening of the circular parental deoxyribonucleic acid (DNA), i.e., conversion of component I to component II of SV40 DNA, takes place after uncoating, followed by the appearance of a new component sedimenting faster than component I at alkaline pH.