Structure of the RNA in tobacco mosaic virus

A model of the RNA of tobacco mosaic virus has been built using computer model-building techniques. The model has good stereochemistry, and fits the electron density map of the virus obtained by fiber diffraction methods considerably better than did earlier models. The three sugar rings in the asymmetric unit all have the A (3′-endo) conformation, One of the bases is in the syn conformation, a conformation observed only rarely in nucleic acid structures.     

Quantized incorporation of RNA during assembly of tobacco mosaic virus from protein disks

Reassembly of tobacco mosaic virus from the isolated RNA and protein, supplied as a disk preparation consisting of over 75% as the disk aggregate, has been followed by the protection of the RNA from nuclease digestion. The sizes of the RNA fragments were determined on agarose/acrylamide gels.During the first few minutes the protected RNA is found to be “quantized” into discrete lengths, differing on average by about 50 or 100 nucleotides, corresponding to one or two turns of the virus helix and strongly supporting the hypothesis that elongation in the major direction, towards the 5′-hydroxyl end, is occurring by the direct addition of protein disks. Protected RNA of the full length found in tobacco mosaic virus is visible within six minutes of starting reassembly, and by 30 minutes most of the RNA is fully protected.     

States of aggregation of the dahlemense strain of tobacco mosaic virus protein and their relation to crystal formation.

The aggregation of the protein of the dahlemense strain of tobacco mosaic virus has been studied by electron microscopy and ultracentrifugation. The aggregates formed are similar to those formed by the vulgare strain, although the particular conditions for their formation are often rather different. Helix formation by dialysis of A protein at pH 8 to acid pH is much more efficient if an intermediate step at pH 7 is introduced. The 20 S particle or two-layer disk is stable over a wide range of pH and ionic strength values. There is no tendency to form short stacks of disks at high ionic strength; instead, 30 S particles are formed that correspond to a pair of interlocked disks giving a “figure-of-eight” appearance in electron micrographs. These particles appear to be the “building blocks” of the protein crystal.     

The positions of the N-terminus and residue 68 in tobacco mosaic virus

Specific chemical modifications of the tobacco mosaic virus coat protein lead to new heavy-atom derivatives. They can be used for the determination of phases in the isomorphous replacement method, but more important they are necessary as markers if one wants to trace the polypeptide chain through an electron density map of limited resolution (10 Å). In addition to the positions of two residues known from previous work, two more residues out of the 158 have now been located in three dimensions. The N-terminus is at the outside of the particle (r = 88 Å), and Lys-68 lies at a radius of 72 Å.     

Photosynthetic electron transport in tobacco leaves infected with tobacco mosaic virus

Tobacco ( Nicotiana tabacum L. cv. Samsun) plants inoculated with different strains of tobacco mosaic virus (TMV) inducing mosaic symptoms of widely varying severity were studied with in vivo chlorophyll fluorescence. This method was used to deduce photosynthetic electron transport efficiency in relation to symptom expression. The quantum yields of photosystem II (PS II) electron transport rate were significantly diminished in virus strains inducing loss of chlorophyll. The reduction in young mosaic-diseased leaves appeared to be due in part to a reduction in the fraction of open reaction centers, whereas in older leaves exhibiting less pronounced symptoms the reduction was mainly caused by a reduced efficiency of capture of excitation energy of open PS II reaction centers. Upon infection with any of the five virus strains PS II seemed to be irreversibly damaged in the inoculated leaves and the ones directly above, indicative of a possible increased susceptibility to photoinhibition in these leaves (Somersalo and Krause 1989) even when no symptoms were apparent. Symptom expression did not appear to be related to the influence of the virus on PS II activity, because the severest effects occurred in the inoculated leaves, which either remained symptomless or developed slight yellowing only. This study demonstrates the usefulness or modulated chlorophyll fluorescence measurements for the investigation of plant-virus interactions. It is particularly important when visual symptoms are absent.     

Protection of transgenic tobacco plants expressing bovine pancreatic ribonuclease against tobacco mosaic virus

Transgenic tobacco plants (Nicotiana tabacum cv. SR1) expressing extracellular pancreatic ribonuclease from Bos taurus and characterized by an increased level of ribonuclease activity in leaf extracts were challenged with tobacco mosaic virus. The transgenic plants exhibited a significantly higher level of protection against the virus infection than the control non-transformed plants. The protection was evidenced by the absence (or significant delay) of the appearance of typical mosaic symptoms and the retarded accumulation of infectious virus and viral antigen. These results demonstrate that modulation of extracellular nuclease expression can be efficiently used in promoting protection against viral diseases.     

Homology between the proteins encoded by tobacco mosaic virus and two tricornaviruses

Summary A comparison was made of the amino acid sequences of the proteins encoded by RNAs 1 and 2 of alfalfa mosaic virus (A1MV) and brome mosaic virus (BMV), and the 126K and 183K proteins encoded by tobacco mosaic virus (TMV). Three blocks of extensive homology of about 200 to 350 amino acids each were observed. Two of these blocks are located in the A1MV and BMV RNA 1 encoded proteins and the TMV encoded 126K protein; they are situated at the N-terminus and C-terminus, respectively. The third block is located in the A1MV and BMV RNA 2 encoded proteins and the C-terminal part of the TMV encoded 183K protein. These homologies are discussed with respect to the functional equivalence of these putative replicase proteins and a possible evolutionary connection between A1MV, BMV and TMV.     

A cDNA from tobacco codes for an inhibitor of virus replication (IVR)-like protein

We have shown previously that localization of tobacco mosaic virus (TMV) in tobacco is associated with a ca. 23 kDa protein that inhibits replication of several plant viruses. This protein, named inhibitor of virus replication (IVR), was purified from the medium of TMV-inoculated protoplasts derived from Nicotiana tabacum cv. Samsun NN. IVR was shown to be present also in induced-resistant leaf tissue of N. tabacum cv. Samsun NN. We prepared an expression cDNA library from such induced-resistant tissue and screened it with a polyclonal antibody raised against the IVR protein. A 1016 bp clone (named NC330) containing a 597 bp open reading frame, coding for a 21.6 kDa polypeptide, was isolated. The NC330 clone hybridized with RNA from induced-resistant tissue from N. tabacum cv. Samsun NN but not with RNA from non-induced tissue. Likewise, it did not hybridize with RNA from infected or uninfected tissue of N. tabacum cv. Samsun nn. Similarly, the NC330 cloned probe hybridized with the RT-PCR products from RNA of the induced-resistant tissue only. In Southern blot hybridization the NC330 DNA probe detected several genomic DNA fragments in both N. tabacum cv. Samsun NN and Samsun nn. The size of the DNA fragments differed in Samsun NN and Samsun nn. We suggest that DNA encoding the IVR-like protein is present in resistant and susceptible N. tabacum genotypes, but is expressed only in NN. We have inserted the NC330 into the expression vector pET22b and a 21.6 kDa protein was produced in Escherichia coli that reacted in immunoblots with the IVR antibody. This protein greatly reduced replication of TMV in N. tabacum cv. Samsun nn leaf disk assays.     

烟草花叶病毒复制酶介导抗性的研究进展

在抗病毒植物基因工程中,利用病毒的复制酶基因是一种很有前途的方法。本对烟草花叶病毒（TMV）的基因组结构及其编码的蛋白的功能作了简介,同时较详细地阐述了由TMV复制本科的通读部分、全长复制酶以及突变或缺失的复制酶介导的对病毒抗性的研究进展。     

Studies on biosynthesis of tobacco mosaic virus. VII. Radioactivity of plus and minus strands in different forms of viral RNA after labelling of infected tobacco leaves

Tobacco leaves were labelled with tritiated undine for 30 or 120 minutes at different times after systemic infection with tobacco mosaic virus. RNA was extracted and separated into three fractions: one enriched in RF (replicative form), one enriched in RI (replicative intermediate), and one containing the bulk of single-stranded RNA. Radioactivity in plus strands (viral RNA) and minus strands (complementary RNA) was determined in each fraction by an isotope dilution assay. The amount of minus strands in the RP and RI fractions and the amount of plus strands in the single-stranded RNA fraction were also determined.Minus-strand synthesis was twice as high a few hours after the outbreak of visible symptoms as during the subsequent large accumulation of plus strands. At the early stage of virus production, the specific radioactivity of the minus strands was three- to fourfold that of the total RNA. Later it was about the same as that of the total RNA. As minus strands constitute a constant part of the total RNA at the later stages, this observation suggests that breakdown of minus strands is small.The specific radioactivity of minus strands was the same in corresponding RF and RI fractions. As the turn-over of minus strands appears to be small, a rapid interconversion of the two RNA types is indicated.In RF and RI the radioactivity in plus strands was between 6 and 50 times greater than that in minus strands. The specific radioactivity of plus strands was greater in RF and RI than in the single-stranded RNA, supporting the concept that both RF and RI have a precursor role for viral RNA.     

Structural comparisons of the aggregates of tobacco mosaic virus protein

The coat protein of tobacco mosaic virus forms numerous aggregates, including the small A-protein, the disk, and two helical forms. The structures of the disk, the helical protein forms, and the virus are compared. Most of the differences are in the conformation of the chain between residues 89 and 113, which lies in the region of protein at the center of the virus, inside the RNA. It is disordered in the disk, but has a fixed conformation in the virus and the protein helices. The differences between the virus and the two helical protein forms are largely in the conformations of arginines and carboxylic acids in this region.     

Kinetics of thermal aggregation of tobacco mosaic virus coat protein

The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 52°C in a wide range of protein concentrations, [P]₀. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 52°C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to [P]₀ (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 42°C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]₀ and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates {A/A _lim; t} (t is time and A _lim is the limiting value of A at t ). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate-limiting stage is the stage of protein molecule unfolding.     

Effect of chitosan on tobacco mosaic virus(TMV) accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus(TMV) in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV(2 μg/mL) and chitosan(1 mg/mL) were lower in the early period of infection(3 days after inoculation), by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases(proteases, RNases) in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid(PTA)-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles(18 nm in diameter, 300 nm long), these suspensions contained abnormal(swollen, "thin" and "short") virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that "thin" virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.     

Effect of chitosan on tobacco mosaic virus（TMV） accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus（TMV） in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV（2 μg/mL） and chitosan（1 mg/mL） were lower in the early period of infection（3 days after inoculation）, by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases（proteases, RNases） in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid（PTA）-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles（18 nm in diameter, 300 nm long）, these suspensions contained abnormal（swollen, “thin” and “short”） virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.