TMV-RNA

Extracts from 152 plant species, representing 46 different families, were screened for effects on tobacco mosaic virus (TMV) replication in cucumber cotyledons. Twenty species have shown enough activity to warrant further study. Several members of the Caprifoliaceae family increased virus replication. An extract of Lonicera involucrata enlarged the virus lesions in local lesion hosts and produced a thirty fold increase in virus titer, but had no effect on virus replication in systemic hosts. The active material appears to affect the virus defense mechanism of local lesion hosts. An extract of common geranium is an active virus inhibitor. It inactivates TMV and TMV-RNA (ribonucleic acid) in vitro by forming non-infectious complexes. In vivo, it also inhibited starch lesion formation in cucumber cotyledons incited by TMV infection. 

Tannic acid is a strong inhibitor of virus particles in vitro. It inactivated both TMV and TMV-RNA by forming noninfectious complexes (1). TMV-RNA was much more sensitive to inactivation than was whole TMV. It would thus appear that tannic acid could possibly inactivate TMV by reacting with either the protein coat or the RNA core. 

Balias, N., Zakay, N., Sela, I., and Loyter, A., Liposome bearing a quaternary ammonium detergent as an efficient vehicle for functional transfer of TMV-RNA into plant protoplast, Biochimica et Biophysica Acta, 1988, 939, 8-18. 

An in vitro reticulocyte translation assay (Boehringer Mannheim) was modified to determine an inhibition of translation by alkaloids. An assay (total volume 25 jl) contained 2 jl 12.5 x translation mix (Boehringer), 10 pi reticulocyte lysate, 200 mM K-acetate, 1.5 mM Mg-acetate, 0.25 pCi L-[4,5-3H(N)]-leucine, 0.5 pg TMV-RNA (Boehringer) and up to 5 mM alkaloids (buffered to pH 7). The mixture was incubated at 30 °C reactions were terminated after 0, 10, 20, 30 and 40 min. The radiolabeled protein was precipitated by adding 200 pi ice-cold trichloroacetic acid (TCA) (50% w/v) and, after 30 min, filtered through GF 34 filters (Schleicher-Schull), which binds proteins. After washing the filters three times with 50% TCA, they were dried at 85 °C. Radioactivity of the filters was determined in a liquid scintillation counter.19... 

Roberts, P. E., and Paterson, B. M. (1973). Efficient Translation of TMV-RNA and Globin 9S RNA in a Cell-Free System from Commercial Wheat Germ. Proc Natl Acad Sci USA 70 2330. 

By combining the results of calorimetric, hydrodynamic and ORD-CD measurements on TMV-RNA obtained at different ionic strength it has been suggested that conformational changes, most likely as a result of base pairing, are responsible for heat effects observed (393). 

Science 81, 644 (1935). Prepn and characterization of essentially uniform TMV particles Boedtker, Simmons, J. Am. Cfiem. Soc. 80, 2550 ([958) Knight, Biochem. Prepns. 9, 132 (1962). Sequential arrangement of the 158 amino acid residues of the protein subunit Tsugita et al, Proc, Nat. Acad. Sci. USA 46, 1463 (1960). Synthesis of TMV-RNA by cell free extracts from infected tobacco leaves Kim, Wildman, Biochem, Biophys. Res. Commun. 8, 394 (1962) Cochran, Chem. 41 Eng. News 48, 64 (Sept. 17, 1962). Reviews An-derer, Advan. Protein Chem. 18, I (1963) Caspar, ibid. 37 Klug, Caspar, Advan. Virus Res. 7, 233-277 (i960) Lauffer, Stevens, f id. 13, 1 (1968) Reddi, ibid. 17, 51 (1972) L Hirth, K. E. Richards, ibid. 26, 145-199 (1981). 

Figure 3. From Tobacco Mosaic Virus to Hepatitis Delta Virus the 3 -end of the TMV RNA genome contains a tRNA-like domain in which a pseudoknotted three-way junction is present (top) two other drawings of the corresponding secondary structure are shown below the drawing at the left reveals the hidden similarity with the proposed secondary structure of the catalytic core of the Hepatitis Delta Virus shown at the bottom. In HDV, the cleaved phosphodiester is at the 5 -end of helix I, while the loop of hairpin III and the junction between helices I and II are crucial for catalysis. It is presently unknown whether this similarity is purely formal or reflects common biological function and/or origins. Adapted from (21).

Hirai A, Wildman SG, Hirai T. Specilic inhibition of TMV-RNA synthesis by bla icidin S. Virology 1968 36 646-651. 

In experiments in vitro TMV-RNA can also stimulate synthesis of polypeptides, even if a synthetic system of ribosomes and supernatant fraction of homogenates of E. coli is used (Tsugita, Fraenkel-Conrat et al., 1962). Some of the products of this synthesis (about 10%) were identical in antigenic and biochemical properties with TMV protein. The rest of the products of synthesis were similar to TMV protein only in certain respects. 

Autoreplication of TMV-RNA takes place in accordance with a principle common to the synthesis of nucleic acids, i.e., by the preliminary formation of complementary forms of RNA (Shipp and Haselkorn, 1964 Burdon, Billeter, et al., 1964 Ralph et al., 1965 Erikson and Franklin, 1966). The intermediate form of this repli-... 

A typical virus with helical symmetry is the tobacco mosaic virus (TMV). This is an RNA virus in which the 2130 identical protein subunits (each 158 amino acids in length) are arranged in a helix. In TMV, the helix has 16 1/2 subunits per turn and the overall dimensions of the virus particle are 18 X 300 nm. The lengths of helical viruses are determined by the length of the nucleic acid, but the width of the helical virus particle is determined by the size and packing of the protein subunits. 

Completely different mechanisms are involved in the self-assembly of the tobacco mosaic virus (TMV). This virus consists of single-strand RNA, which is surrounded by 2,130 identical protein units, each of which consists of 158 amino acid residues. A virus particle, which requires the tobacco plant as a host, has a rodlike structure with helical symmetry ( Stanley needles ). It is 300 nm long, with a diameter of 18nm. The protein and RNA fractions can be separated, and the viral... 

The majority of viruses that infect plants have single-stranded, positive-sense RNA genomes. It has therefore been necessary to use infectious cDNA clones for the in vitro manipulation of RNA viruses, allowing them to be developed as effective tools for the commercial production of target proteins in plants. This approach has also been used to study the genetic and metabolic profiles of both viruses and their host plants. Siegel [14] conceptualized the potential use of RNA viruses as expression vectors. Brome mosaic virus (BMV) and Tobacco mosaic vims (TMV) were the first two RNA viruses to be converted into expression vectors. These vectors have since been pro-... 

True self-assembly is observed in the formation of many oligomeric proteins. Indeed, Friedman and Beychok reviewed efforts to define the subunit assembly and reconstitution pathways in multisubunit proteins, and all of the several dozen examples cited in their review represent true self-assembly. Polymeric species are also formed by true self-assembly, and the G-actin to F-actin transition is an excellent example. By contrast, there are strong indications that ribosomal RNA species play a central role in specifying the pathway to and the structure of ribosome particles. And it is interesting to note that the assembly of the tobacco mosaic virus (TMV) appears to be a two-step hybrid mechanism the coat protein subunits first combine to form 34-subunit disks by true self-assembly from monomeric and trimeric com-... 

FIGURE 4.2 Schematic diagram of RNA virus expression vectors, (a) TMV as an epitope presentation system, and (b) a polypeptide presentation system. Dark diamonds represent foreign antigen/peptide. 

Certain viruses, notably tobacco mosaic virus (TMV), can also self-assemble. A TMV particle can be dissociated into its component proteins and RNA and then reassembled into infective virus particles on mixing the components together again. 

TMV consists of a cylindrical coat of 2,130 identical protein subunits enclosing a long RNA molecule of 6,400 nucleotides. In 1955, it was shown that the coat protein subunits and the RNA could be dissociated but would, under appropriate conditions, spontaneously self-assemble to reform fully active virus particles. This process is multistage, the critical intermediate being a 34-unit two-layered protein disc which, upon binding the RNA, is converted to a helical structure with 16.33 protein subunits per turn (Fig. 5-2). In the absence of the RNA, the protein may be polymerized into helical tubes of indefinite length. The presence of the RNA aids the polymerization process and results in a virus particle with a fixed length of 300 nm. 

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