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PubMed1984

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.

Adenine Nucleotides↗
PubMed1996

Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase.

A crude membrane-bound RNA polymerase, obtained by differential centrifugation of extracts of tomato leaves infected with tobacco mosaic tobamovirus (tomato strain L) TMV-L), was purified by sucrose density gradient centrifugation. Removal of the endogenous RNA template with micrococcal nuclease rendered the polymerase template dependent and template specific. The polymerase was primer independent and able to initiate RNA synthesis on templates containing the 3'-terminal sequences of the TMV-L positive or negative strands. TMV-vulgare RNA was a less efficient template, while RNAs of cucumber mosaic cucumovirus and red clover necrotic mosaic dianthovirus, or 5'-terminal sequences of TMV-L positive or negative strands, did not act as templates for the polymerase. A main product of the reaction with TMV-L genomic RNA as a template, carried out in the presence of [alpha-32P]UTP, was genomic-length single-stranded RNA. This was shown to be the positive strand and uniformly labelled along its length, demonstrating complete replication of TMV-L RNA. Genomic-length double-stranded RNA, labelled in both strands, and small amounts of RNAs corresponding to the single- and double-stranded forms of the coat protein subgenomic mRNA were also formed. Antibodies to N-terminal and C-terminal portions of the 126-kDa protein detected the 126-kDa protein and the 183-kDa readthrough protein in purified RNA polymerase preparations, whereas antibodies to the readthrough portion of the 183-kDa protein detected only the 183-kDa protein. All three antibodies inhibited the template-dependent RNA polymerase, but none of them had any effect on the template-bound enzyme.

Antibodies↗
PubMed1997

The tobacco mosaic virus RNA polymerase complex contains a plant protein related to the RNA-binding subunit of yeast eIF-3.

A sucrose density gradient-purified, membrane-bound tobacco mosaic virus (tomato strain L) (TMV-L) RNA polymerase containing endogenous RNA template was efficiently solubilized with sodium taurodeoxycholate. Solubilization resulted in an increase in the synthesis of positive-strand, 6.4-kb genome-length single-stranded RNA (ssRNA) and a decrease in the production of 6.4-kbp double-stranded RNA (dsRNA) to levels close to the limits of detection. The solubilized TMV-L RNA polymerase was purified by chromatography on columns of DEAE-Bio-Gel and High Q. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed that purified RNA polymerase preparations consistently contained proteins with molecular masses of 183, 126, 56, 54, and 50 kDa, which were not found in equivalent material from healthy plants. Western blotting showed that the two largest of these proteins are the TMV-L-encoded 183- and 126-kDa replication proteins and that the 56-kDa protein is related to the 54.6-kDa GCD10 protein, the RNA-binding subunit of yeast eIF-3. The 126-, 183-, and 56-kDa proteins were coimmunoaffinity selected by antibodies against the TMV-L 126-kDa protein and by antibodies against the GCD10 protein. Antibody-linked polymerase assays showed that active TMV-L RNA polymerase bound to antibodies against the TMV-L 126-kDa protein and to antibodies against the GCD10 protein. Synthesis of genome-length ssRNA and dsRNA by a template-dependent, membrane-bound RNA polymerase was inhibited by antibodies against the GCD10 protein, and this inhibition was reversed by prior addition of GCD10 protein.

DNA-Directed RNA Polymerases↗
PubMed2005

Interaction of the tobacco mosaic virus replicase protein with the Aux/IAA protein PAP1/IAA26 is associated with disease development.

Virus-infected plants often display developmental abnormalities that include stunting, leaf curling, and the loss of apical dominance. In this study, the helicase domain of the Tobacco mosaic virus (TMV) 126- and/or 183-kDa replicase protein(s) was found to interact with the Arabidopsis Aux/IAA protein PAP1 (also named IAA26), a putative regulator of auxin response genes involved in plant development. To investigate the role of this interaction in the display of symptoms, a TMV mutant defective in the PAP1 interaction was identified. This mutant replicated and moved normally in Arabidopsis but induced attenuated developmental symptoms. Additionally, transgenic plants in which the accumulation of PAP1 mRNA was silenced exhibit symptoms like those of virus-infected plants. In uninfected tissues, ectopically expressed PAP1 accumulated and localized to the nucleus. However, in TMV-infected tissues, PAP1 failed to accumulate to significant levels and did not localize to the nucleus, suggesting that interaction with the TMV replicase protein disrupts PAP1 localization. The consequences of this interaction would affect PAP1's putative function as a transcriptional regulator of auxin response genes. This is supported by gene expression data indicating that approximately 30% of the Arabidopsis genes displaying transcriptional alterations in response to TMV contain multiple auxin response promoter elements. Combined, these data indicate that the TMV replicase protein interferes with the plant's auxin response system to induce specific disease symptoms.

Arabidopsis Proteins↗
PubMed2004

Tobacco mosaic virus: a model system for plant biology.

Tobacco mosaic virus (TMV) has had an illustrious history for more than 100 years, dating to Beijerinck's description of the mosaic disease of tobacco as a contagium vivum fluidum and the modern usage of the word "virus." Since then, TMV has been acknowledged as a preferred didactic model and a symbolic model to illuminate the essential features that define a virus. TMV additionally emerged as a prototypic model to investigate the biology of host plants, namely tobacco. TMV also exemplifies how a model system furthers novel, and often unexpected, developments in biology and virology. Today, TMV is used as a tool to study host-pathogen interactions and cellular trafficking, and as a technology to express valuable pharmaceutical proteins in tobacco. The history of TMV illustrates how pragmatic strategies to control an economically important disease of tobacco have had unexpected and transforming effects across platforms that impinge on plant health and public health.

Agriculture↗
PubMed1998

Budesonide affects allergic mucociliary dysfunction.

Airway inflammation characterized by neutrophils and free elastase contributes to allergic mucociliary dysfunction. Glucocorticosteroids are the most important anti-inflammatory agents used in the treatment of asthma, but their effect on allergic mucociliary dysfunction is not known. Therefore, we assessed both the prophylactic and therapeutic effects of the glucocorticosteroid budesonide on antigen-induced mucociliary dysfunction in sheep. Tracheal mucus velocity (TMV), a marker of mucociliary clearance, was measured by using a roentgenographic technique. When budesonide was administered either 30 min before or 1 h after airway challenge with Ascaris suum, the antigen-induced fall in TMV at 6 h was prevented. The effects on TMV at 8 and 24 h after challenge were also determined when budesonide and, for comparative purposes, alpha1-protease inhibitor were given 6 h after antigen challenge. Budesonide treatment improved TMV at 8 h, but TMV was not significantly different from antigen alone at 24 h. Treatment with alpha1-protease inhibitor, however, caused only a significant reversal of the antigen-induced fall in TMV at 24 h after challenge; this indicates a more prolonged effect than budesonide. Our results suggest that antiproteases may have a potential role as a therapeutic approach to mucociliary dysfunction in asthma and provide evidence for another means by which glucocorticosteroids contribute to the control of the disease.

Aerosols↗
PubMed1978

Recovery of tobacco mosaic virus RNA Replication after incubation at 40 degrees.

Incubation of tobacco mosaic virus (TMV)-infected tissue at 40 degrees for 12 h destroyed the capacity for TMV RNA replication. Upon shift of the tissue from 40 to 25 degrees, synthesis of TMV RNA did not resume. Incubation at 40 degrees for 1 h did not destory the TMV RNA replicase, because viral RNA synthesis resumed upon return to 25 degrees. However, upon further incubation at 25 degrees, the synthesis rate of TMV RNA gradually declined. Upon still further incubation at 25 degrees (16-20 h), the synthesis of TMV RNA in tissue incubated at 40 degrees for either 1 or 12 h recovered. This recovery was inhibited by cycloheximide but not by 2-thiouracil.

Cycloheximide↗
PubMed1992

Impairment of airway mucociliary transport by Pseudomonas aeruginosa products. Role of oxygen radicals.

We previously showed that the supernatant of a Pseudomonas aeruginosa (PA) culture and its constituents pyocyanin and 1-hydroxyphenazine inhibit ciliary activity of dispersed tracheal epithelial cells in vitro via the generation of oxygen radicals by phagocytes. In the present study, we wished to determine if tracheal mucus velocity (TMV) is also impaired by PA supernatant and if oxygen radicals have a mediating role. In conscious sheep, TMV (measured with a radiographic method) was determined before and serially following aerosol challenge with the cell-free supernatant of a PA culture or unconditioned culture medium (control). TMV decreased from a mean (+/- SEM) baseline of 6.7 +/- 1.1 mm/min (n = 6) by 29, 35, and 25% at 0.5, 3, and 24 h after challenge, respectively (p < 0.05), and returned to baseline 1 wk later (-6%, p = NS). Control medium had no effect on TMV (maximum decrease by 15% at 0.5 h). Aerosolized catalase blunted the effect of PA supernatant on TMV. To determine if the impairment of TMV involved ciliary inhibition, tissues were mounted in a chamber and ciliary beat frequency (CBF) and surface liquid velocity (SLV) were measured with a microscopic method. PA supernatant decreased both CBF (maximum mean decrease 12%; n = 5, p < 0.05) and SLV (maximum mean decrease 78%; n = 5, p < 0.05) in a dose-dependent fashion, with a correlation between the two parameters; these effects were blocked by catalase.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed2004

The symptom difference induced by tobacco mosaic virus and tomato mosaic virus in tobacco plants containing the N gene is determined by movement protein gene.

Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.

Plant Diseases↗
PubMed1989

Translational capacity of sheep oocytes microinjected with messenger RNA.

Sheep oocytes were microinjected with tobacco mosaic virus RNA (TMV-RNA) and isotopically labelled with L-[35S]methionine. Total incorporation of labelled methionine was similar in TMV-RNA-injected and in carrier-injected control oocytes, whether injections were performed during the period of high protein synthesis at maturation or during the period of reduced synthesis at a time equivalent to the mid-cleavage transition (48 h after germinal vesicle breakdown). Varying the amount of TMV-RNA injected from 2.5 to 10 pg had little effect on the overall level of amino acid incorporation. Furthermore TMV-RNA appeared to be very stable in oocytes and eggs; the proportion of total polypeptide synthesis directed by TMV-RNA did not diminish during the first 48 h after injection. Synthesis of most endogenous proteins was uniformly reduced to compensate for the synthesis of TMV-polypeptides. Our results suggest, therefore, that the translational capacity of sheep oocytes is fully saturated during maturation.

Animals↗
PubMed1999

Genetically engineered transgenic plants with the domain 1 sequence of tobacco mosaic virus 126 kDa protein gene are completely resistant to viral infection.

In many plant RNA viruses, Domains 1, 2 and 3 are conserved in replicase proteins. In order to examine the interference of viral replication by the Domain 1 sequence, we generated transgenic plants transformed with DNA corresponding to the Domain 1 sequence of the TMV 126 kDa protein. This DNA sequence includes the TMV RNA from nucleotides 1 to 2,149, which comprises both the 5'-untranslated and methyl transferase region. The transgenic plants obtained showed complete resistance to TMV infection. The presence of the Domain 1 sequence in the plants completely prevented local necrosis in Nicotiana tabacum cv. Xanthi nc, and any systemic development of symptoms in Nicotiana tabacum Xanthi upon TMV inoculation. Most transgenic plants sustained the conferred resistance even under TMV inoculum concentrations up to as high as 1,000 microg/ml. To detect any accumulation of TMV coat protein or viral RNA in infected transgenic plants, immunochemical tests and Northern blot analyses were carried out. Neither viral RNA or coat protein was detectable in the systemic leaves of the completely resistant transgenic plants, whereas they were accumulated in large quantities in all of the control plants. Because of the conservation of Domain 1 in many plant RNA viruses, the acquisition of resistance to virus infection using the Domain 1 sequence appears to be a very effective strategy for breeding of viral resistant plants.

Genes, Viral↗
PubMed1975

Assembly of tobacco mosaic virus in vitro. Improved model for the elongation process by protein subunits.

The in vitro assembly reaction of tobacco mosaic virus (TMV), especially the elongation process of partially reconstituted RNA (PRR) by protein subunits, was observed by electron microscopy. After addition of TMV-protein subunits, the PRR appeared as rods with a clump at one end, believed to be a complex between added protein subunits and the RNA tail protruding from PRR. The subunits entrapped on the RNA tails in the forms of clumps were progressively incorporated into the growing rods on incubation, ending with the formation of completely reconstituted rods. The clumps were also observed after addition of cucumber green mottle mosaic virus (CGMMV) protein subunits to rods partially reconstituted from RNA and TMV-protein. In this case, the protein subunits, seen as clumps, did not become incorporated to form elongating rods. An improved model for the elongation of TMV rods is proposed. The elongation process is composed of two steps, with the first step being the interaction of protein subunits with the RNA tail protruding from the growing rod. Any protein having a specific binding site for TMV-rna, not limited to TMV-protein, will react in the first step. The second step is the incorporation of the protein on the RNA tail into a rod-shaped structure, with consequent elongation of the growing rod. It appears that only protein homologous with that in the partially reconstituted rods can partake in the second step.

Microscopy, Electron↗
PubMed2004

Experimental and natural weed host-virus relations.

Weeds, as alternative hosts of plant viruses and nutrient plants of virus vectors play important role in virus ecology and epidemiology. The aim of our study was to discover new weed-virus relations. Therefore some weed species were mechanically inoculated with 28 viruses (strains or isolates) maintained in our glasshouse. Different weed species with and without visible symptoms were collected from agro-, water ecosystems and wastelands of Hungary between 1997 and 2003. Virus infections were evaluated by biotests, DAS ELISA serological methods, electronmicroscopy and immunosorbent electronmicroscopy (ISEM). Under glasshouse conditions Ambrosia artemisifolia was considered as a virophob species, showing resistance to all viruses listed above. A series of new artificial (Chenopodium album--SoMV (LH+SH)*, AMV (LH+SH); C. berlandieri--PVY(NTN) (LH), AMV (LH+SH), CMV (LH), SoMV (LH+SH), ObPV (LH+SH), ZYMV-10 (LH): C. ugandae--ObPV (LH), SoMV (L); C. glaucum--ObPV (LH), SoMV (L); Echinocystis lobata--PVX (L), ZYMV (LH+SH); Solanum nigrum--MYFV (LH+SH), PVY(N) (L), PVY(NTN) (LH+SH), SoMV (LH), TMV (SH), CMV (SH); S. dulcamara--CMV-U/246 (SH), PVY(NTN) (LH), SoMV-H (L), TMV-O (L); S. luteum--PVY(N) (SH), PVY(NTN) (LH+L), TMV(SH).) and natural (Asclepias syriaca--TMV, AMV, TSWV; Alisma plantago-aquatica--PVY, SoMV; Ambrosia artemisiifolia--CMV; Chenopodium album--CMV, PVS, PLRV; C. hybridum--CMV; Cirsium canum--CMV, PVM; Carex vulpina--CMV; Comium maculatum--PVY; Datura stramonium--PVA, PVX, PVS, PVM, CMV, TMV; Lysimachia vulgaris--ArMV, BNYVV, CMV, TMV; Lythrum salicaria--ArMV; Malva neglecta--CMV; Mercurialis annua--SoMV; Solanum nigrum--CMV, PVY, PVY(N); Solidago gigantea--CMV, RpRSV, BNYVV; Stenactis annua--PVM, PVA) weed--virus relations were detected. The epidemiological role of perennial hosts (A. syriaca, A. planlago aquatica, C. canurm, L. vulgaris, L. salicaria, S. gigantea) is especially high, because they can serve as infection sources as well as overwintering hosts of different plant viruses.

Antiviral Agents↗
PubMed2005

[A single reverse mutation in the 126/183-kda replicase gene of the attenuated tomato strain V-69 of tobacco mosaic virus increases the virus pathogenicity].

To determine the roles of the mutations that were earlier found in the structure of the attenuated tomato strain V-69 of tobacco mosaic virus (TMV), the nucleotide sequences of genomic RNAs of its four pathogenic revertants have been identified and analyzed. The comparison of the structures of viral genomes has demonstrated that all revertants studied have a single reverse mutation at position 1654 relative to the parental TMV strain V-69. This reversion has been introduced into the plasmid construction harboring a full-length cDNA copy of the TMV strain V-69 RNA, and the infective transcripts synthesized in vitro have been tested on TMV-sensitive tomato and tobacco plants. The tests have confirmed that the single reversion at position 1654, which changes the amino acid residue at position 528 of the 126-kDa and 183-kDa proteins of the viral replicase is responsible for the increase in the pathogenicity of revertants compared to the TMV V-69 strain. The mutation at position 1654 of the viral genome may be supposed to be the main factor of the attenuation of TMV strain V-69.

Base Sequence↗
PubMed1989

[Identification of a peptide of the membrane protein of tobacco mosaic virus, cross-linked with intraviral RNA under the effect of UV-radiation].

As reported previously, UV-irradiation induces crosslinking between tobacco mosaic virus (TMV) coat protein molecules and intraviral RNA nucleotides. We have irradiated [3H]-uridine labeled TMV and isolated TMV coat protein subunits with the attached nucleotide label. These TMV protein subunits were hydrolyzed with trypsin. The tryptic peptides were separated by high-performance liquid chromatography and [3H]-labeled peptides were identified. The UV-irradiation of TMV was found to result in crosslinking to intraviral RNA of the T8 tryptic peptide (residues 93-112) of TMV coat protein.

Capsid↗
PubMed1986

Impairment of tracheal mucociliary clearance but not ciliary beat frequency by a combination of low level ozone and sulfur dioxide in sheep.

We used conscious sheep to determine the effect of a combined exposure to ozone (O3) and sulfur dioxide (SO2) on tracheal mucus velocity (TMV) and ciliary beat frequency (CBF). TMV was measured in vivo by tracking the movement of radiopaque particles which had been deposited on the tracheal mucosa. CBF of tracheal epithelial cells, obtained by brushing, was measured in vitro with phase contrast microscopy. On two separate occasions, six sheep were exposed by mask to either a combination of 0.3 ppm O3 and 3 ppm SO2 or air (control) for 5 h on each of three consecutive days. TMV and CBF were measured prior to the first exposure, and immediately and 24 h after the last exposure. The combination of O3 and SO2 depressed mean TMV by 40% immediately after and by 25%, 24 h after exposure. Both values were different from the corresponding values after air exposure (p less than 0.05). However, this depression in TMV was not associated with an impairment in CBF. We conclude that in conscious sheep, exposure to a combination of low level O3 and SO2 causes a depression in TMV, and changes in CBF are probably not a primary cause of this mucociliary dysfunction.

Animals↗
PubMed1980

Differential pulse-polarographic analysis of tobacco mosaic virus.

The tobacco mosaic virus (TMV) strain vulgare and its mutant TMV 483 (with glutamine-9 replaced by histidine) and the denatured protein of TMV vulgare were analysed by direct current (d. c.) and differential (derivative) pulse polarography (DPP) in the basic electrolyte composed of 0.001 M Co(NH3)6Cl3, 0.1 M NH4Cl and 0.1 M NH3 at 0 degrees C. The DPP method gave a substantially better resolution of the polarographic catalytic maxima A and B, but a much lower resolution of the maxima B and C, as compared with the d. c. polarographic method. The clear differentiation of the maximum A from maximum B by DPP permitted to study the variation of maximum A in the course of alkaline degradation of TMV. But for the study of TMV protein denaturation the d. c. polarography is preferable, because the denaturation is accompanied by the appearance and rise of maximum C which can be clearly differentiated from maximum B by d. c. polarography rather than by DPP. The DPP method was more, sensitive than d. c. polarography. The denatured TMV protein can be determined by DPP at concentrations around 0.1 microgram/ml.

Histidine↗
PubMedDate not supplied

[Directed fragmentation of tobacco mosaic virus RNA: excision of 3' terminal sections, containing coat protein genes of tobacco mosaic virus].

The present work demonstrated that RNA of different Tobacco mosaic virus strains (common; A14; Ni118, U2; wheat strain) are selectively cleaved by RNase H from E. coli in the presence of oligodeoxyribonucleotide d(TTTTTTTCCGG) complementary to the stretch of TMV RNA from the 842-nd to the 852-nd nucleotide from 3'-end, e. g. corresponding to the region adjacent to the 3'-terminus of origin of assemble of TMV. Such cleavage yields two main products. One of them--the short S-fragment--is the 3'-terminal part of TMV RNA. This conclusion is based on its ability to accept histidine and its molecular weight is about 0.28 X 10(6). The other main product of TMV RNA cleavage by RNase H is the L-fragment. L-fragment is the 5'-terminal moiety of TMV RNA, as follows from its ability to direct the in vitro synthesis of p110-130, which is known to be encoded in the 5'-terminal TMV RNA region.

Antigens, Viral↗