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.     

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.     

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.     

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。     

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.     

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.     

Isolation and characterization of basil mosaic virus in Egypt

Different viral infection symptoms were observed on leaves of basil plants (Ocimum basilicum L.), growing at El-Monofia Governorate, Egypt, during 2015–2016. The majority of these symptoms were mosaic and leaf malformation. The pathogenic agent of this disease is known as basil mosaic virus (BsMV). The sensitive hosts to BsMV are Chenopodium amaranticolor and Phaseolus vulgaris cv. (brown beans). The biological properties of BsMV were found to be 10^?4, 6 days and 66 °C for dilution end point, longevity in vitro and thermal inactivation point, respectively. The electron micrograph of the isolated virus indicated this virus was found to be isometric particles of approximately, 50 nm in diameter and could be classified as tentative member of the Fabavirus. Real-time PCR detection by the Fab primer pairs was confirmed at Ct value = 25.44 for the total cDNA isolated from infected Basil plants and 27.94 for the cDNA of purified virus particles, while CMV primer pairs showed no amplification for both samples. According to the available reports this seems to be the first record for basil mosaic virus in Egypt.     

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.     

Complete nucleotide sequence and genome organization of tobacco mosaic virus isolated from Viciafaba

Based on reported TMV-U1 sequence, primers were designed and fragments covering the entire genome of TMV broad bean strain (TMV-B) were obtained with RT-PCR. These fragments were cloned and sequenced and the 5' and 3' end sequences of genome were confirmed with RACE. The complete sequence of TMV-B comprises 6 395 nucleotides (nt) and four open reading frames, which correspond to 126 ku (1 116 amino acids), 183 ku (1 616 amino acids), 30 ku (268 amino acids) and 17.5 ku proteins (159 amino acids). The complete nucleotide sequence of TMV-B is 99.4% identical to that of TMV-U1. The two virus isolates share the same sequence of 5', 3' non-coding region and 17.5 K ORF, and 6, 1 and 3 amino acid changes are found in 126 K protein, 54 K protein and 30 K protein, respectively. The possible mechanism on the infection of TMV-B in Vicia faba is discussed.     

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 ?-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 ?-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.     

An analysis of tobacco mosaic virus replicative structures synthesized in vitro

Summary The RNA structures synthesized in vitro by a crude enzyme complex from tobacco mosaic virus (TMV)-infected leaves have been analyzed; the major viral-specific products were similar to TMV-replicative form (RF) and-replicative intermediate (RI) in electrophoretic behavior and ribonuclease sensitivity. Synthesis of these RF-like and RI-like structures neither required nor responded to added viral RNA, but did require all four ribonucleotide triphosphates. Enriched radiolabeled RF-like and RI-like RNA fractions were isolated from non-denaturing agarose gels by electroelution and hybridized to a collection of TMV sequences cloned into bacteriophage M13. Enriched RF-RNA hybridized to sequences of both plus and minus polarity, while enriched RI-RNA hybridized only to inserts of minus polarity, indicating only plus strand synthesis in this fraction. Most of the label incorporated into the plus strand of the enriched RF-RNA was found near the 3-end of this strand, while most of the label incorporated into enriched RI-RNA was found several hundred bases from the 5-end of the plus strand.Paper presented at the first International Congress of Plant Molecular Biology (Savannah, GA, 1985).     

TOM1 family conservation within the plant kingdom for tobacco mosaic virus accumulation

The susceptibility factor TOBAMOVIRUS MULTIPLICATION 1 (TOM1) is required for efficient multiplication of tobacco mosaic virus (TMV). Although some phylogenetic and functional analyses of the TOM1 family members have been conducted, a comprehensive analysis of the TOM1 homologues based on phylogeny from the most ancient to the youngest representatives within the plant kingdom, analysis of support for tobamovirus accumulation and interaction with other host and viral proteins has not been reported. In this study, using Nicotiana benthamiana and TMV as a model system, we functionally characterized the TOM1 homologues from N. benthamiana and other plant species from different plant lineages. We modified a multiplex genome editing tool and generated a sextuple mutant in which TMV multiplication was dramatically inhibited. We showed that TOM1 homologues from N. benthamiana exhibited variable capacities to support TMV multiplication. Evolutionary analysis revealed that the TOM1 family is restricted to the plant kingdom and probably originated in the Chlorophyta division, suggesting an ancient origin of the TOM1 family. We found that the TOM1 family acquired the ability to promote TMV multiplication after the divergence of moss and spikemoss. Moreover, the capacity of TOM1 orthologues from different plant species to promote TMV multiplication and the interactions between TOM1 and TOM2A and between TOM1 and TMV-encoded replication proteins are highly conserved, suggesting a conserved nature of the TOM2A–TOM1–TMV Hel module in promoting TMV multiplication. Our study not only revealed a conserved nature of a gene module to promote tobamovirus multiplication, but also provides a valuable strategy for TMV-resistant crop development.     

Glutinosone,a new antifungal sesquiterpene from Nicotiana glutinosa infected with tobacco mosaic virus

Glutinosone is an antifungal substance induced in leaves of Nicotiana glutinosa following infection with tobacco mosaic virus. From chemical, spectroscopic and biogenetic evidence it has been assigned structure (1).