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PubMed2006

Direct interaction between the tobacco mosaic virus helicase domain and the ATP-bound resistance protein, N factor during the hypersensitive response in tobacco plants.

Plants cope with pathogens with distinct mechanisms. One example is a gene-for-gene system, in which plants recognize the pathogen molecule by specified protein(s), this being called the R factor. However, mechanisms of interaction between proteins from the host and the pathogen are not completely understood. Here, we analyzed the mode of interaction between the N factor, a tobacco R factor, and the helicase domain (p50) of tobacco mosaic virus (TMV). To this end, domain dissected proteins were prepared and subjected to Agroinfiltration into intact leaves, followed by yeast two hybrid and pull-down assays. The results pointed to three novel features. First, the N factor was found to directly bind to the p50 of TMV, second, ATP was pre-requisite for this interaction, with formation of an ATP/N factor complex, and third, the N factor was shown to possess ATPase activity, which is enhanced by the p50. Moreover, we found that intra- and/or inter-molecular interactions take place in the N factor molecule. This interaction required ATP, and was disrupted by the p50. Based on these results, we propose a following model for the TMV recognition mechanism in tobacco plants. The N factor forms a complex with ATP, to which the helicase domain interacts, and enhances ATP hydrolysis. The resulting ADP/N factor complex then changes its conformation, thereby facilitating further interaction with the down-stream signaling factor(s). This model is consistent with the idea of 'protein machine'.

Adenosine Triphosphate↗
PubMedDate not supplied

[Epitope mapping of the recombinant movement protein of the tobacco mosaic virus using monoclonal antibodies].

The movement protein (MP) of the tobacco mosaic virus (TMV) provides the intercellular transport of the viral RNA through plasmodesmata. The MP fulfills its function while interacting with host cell factors over the whole path of its intracellular movement from the subcellular site of its synthesis to the plasmodesmata of cellular walls. The MP conformation during its intracellular movement and fulfillment of the transport function still remains unknown. In this study, we describe the preparation of murine monoclonal antibodies (MAs) to TMV MP and mapping of the MP epitopes. Stable hybridoma lines that produce MAs to the partially denatured recombinant MP (MPr) were obtained. MAs were tested by immunoblotting and ELISA with the use of deletion variants of MPr. The epitopes of TMV MPr that recognize specific MAs were determined.

Animals↗
PubMed1984

Immunogold for detection of antigen on nitrocellulose paper.

The application of immunogold for the detection of antigen-antibody complexes on nitrocellulose paper is described. Tobacco mosaic virus (TMV) protein, either purified or in leaf extract, was bound to the nitrocellulose paper and then exposed to rabbit anti-TMV serum. The antigen-antibody complexes were detected by gold-labeled goat anti-rabbit immunoglobulin G. The gold-labeled antibody is directly visible because of its pink color. This method can detect 1-5 pg of TMV protein, either in purified form or in the unpurified plant extract, with high specificity.

Animals↗
PubMed1991

Interaction of non-enveloped plant viruses and their viral coat proteins with phospholipid vesicles.

The interaction of the non-enveloped plant viruses TMV (rod-shaped) and CCMV (spherical) and of their coat proteins in several well-defined aggregation states, with artificial membranes was investigated to study the early stages of the cellular infection process. Information about the separate steps in the interaction mechanisms was obtained by employing three assays, performed as a function of vesicle size, net membrane charge, pH and ionic strength. The assays allow to discriminate between aggregation of vesicles (turbidity assay) and membrane destabilization (vesicle leakage assay and lipid mixing assay). The aggregation of the vesicles is a result of electrostatic interactions between the viral material and vesicles surface (cross-linking), while the destabilization of the membrane is a result of penetration or bilayer disruption by hydrophobic protein domains. TMV virions and its coat protein, and CCMV virions, due to their net negative charge, predominantly interact with positively charged membranes. The coat protein of CCMV was found to interact with negatively charged membranes, an interaction that can be assigned to its basical N-terminal sequence. Changing the aggregational state of the viral coat proteins yielded most significant interactions in case of TMV coat protein aggregated in the disk form and CCMV coat protein aggregated in empty capsids with oppositely charged membranes. These protein aggregates are found to be the best compromise between efficiency (capacity of the protein to bridge vesicles and destabilize their membranes) and concentration of protein aggregates. The results are discussed with respect to previously proposed biological models of the early stages of plant virus infection.

Capsid↗
PubMed1981

Degradation of nucleic acids with ozone. II. Degradation of yeast RNA, yeast phenylalanine tRNA and tobacco mosaic virus RNA.

The degradation of a mixture of four 5'-ribonucleotides (AMP, GMP, CMP and UMP), yeast RNA, yeast phenylalanine tRNA, and tobacco mosaic virus RNA (TMV-RNA) with ozone (concentration in inlet gas, 0.1-0.5 mg/l) was examined in a phosphate buffer (pH 6.9). In the case of the mixture, GMP alone was degraded in the initial stage. In the ozonization of yeast RNA, the guanine moiety was less vulnerable to attack by ozone than in the case of free GMP, but it again degraded most rapidly among the four nucleotides. In the treatment of tRNA with ozone, the guanine moiety degraded first. When the numbers of degraded nucleotides reached 4.8 (remaining amino acid acceptor activity was 3.6%), the polyacrylamide gel electrophoresis of the ozonized tRNA gave a single band with the same mobility as that of the intact tRNA. It is evident that ozonolysis of tRNA proceeded without cleavage of the polynucleotide chain. In the case of TMV-RNA, the loss of the infectivity by ozone proceeded rapidly within 30 min and was followed by preferential degradation of the guanine moiety. The outstanding lability of the guanine moiety observed in each case is discussed in connection with the inactivation of tRNA and TMV-RNA.

Chemical Phenomena↗
PubMed1971

Circular dichroism studies on coat proteins of some strains and mutants of tobacco mosaic virus.

1. Tobacco mosaic virus (TMV) protein has in near ultraviolet a complex but well resolved circular dichroism (CD) spectrum at room temperature. There are seven positive bands at 248, 252, 257, 265, 274, 281 and 291 nm, and a negative one at 296 nm. The CD spectrum is pH-dependent. The shape of the pH-dependence curves and the comparison with CD spectra of model compounds suggest that the bands at 248, 252 and 257 nm are mainly caused by phenylalanyl, those at 265, 274 and 281 nm by tyrosyl, and those at 291 and 296 nm by tryptophanyl side chains. 2. Only insignificant changes of the tertiary structure seem to occur between pH 6.5 and 8.5. Changes in ellipticity of TMV protein during the pH-induced polymerization reaction suggest that: (1) tyrosyl residues are involved in the binding of subunits, (2) phenylalanyl residues seem to be transferred to a less rigid environment, and (3) tryptophanyl residues are not essential for the reaction. 3. The proteins of several TMV strains and mutants studied have similar far ultraviolet CD spectra and apparently do not differ significantly in their structure. Their near ultraviolet CD spectra are, however, different. Replacements involving aliphatic amino acids do not change considerably the near ultraviolet CD spectra. On the other hand, replacements involving aromatic amino acids have a great effect on the spectra rendering possible identification of CD bands and recognition of the aromatic amino acid residues responsible for optical activity.

Amino Acid Sequence↗
PubMed1989

Selective inhibition of photosystem II in spinach by tobacco mosaic virus: an effect of the viral coat protein.

Leaves of Spinacia oleracea inoculated with tobacco mosaic virus (TMV) strain PV230 develop mild chlorotic and mosaic symptoms of infection. Thylakoid membranes isolated from these infected leaves showed a reduced Fv/Fm ratio for chlorophyll fluorescence kinetics, at 25 degrees C. The photosystem II (PS II)-mediated electron-transport rate was inhibited 50%, whereas PS I activity was unaffected by virus infection. Protein analysis indicated that TMV coat protein was associated with thylakoids, in particular with the PS II fraction. The results demonstrate that TMV-infected S. oleracea shows inhibition of photosynthetic electron transport through PS II. We propose that the inhibition of photosynthetic activity results from the association of viral coat protein with the PS II complex.

Blotting, Western↗
PubMed1990

Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector.

To examine the validity of the strategy to express a foreign gene as a fusion protein with the coat protein (CP) of tobacco mosaic virus (TMV), we have constructed ENK RNA by using an in vitro transcription system of TMV RNA. ENK RNA differs from TMV RNA only in that ENK RNA carries an additional sequence coding for Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) (Enk) with a preceding in-frame methionine just before the termination codon of CP gene. In protoplasts inoculated with ENK RNA, CP + Enk fusion protein accumulated as the major protein.

Amino Acid Sequence↗
PubMed1994

The immobilized movement proteins of two tobamoviruses form stable ribonucleoprotein complexes with full-length viral genomic RNA.

The movement proteins of two tobamoviruses (tobacco mosaic virus, TMV, common strain U1 and cruciferous TMV strain) containing amino-terminal hexahistidine affinity tags were overexpressed in Escherichia coli and purified by metal chelate affinity chromatography. Purified recombinant proteins were immobilized to a Ni(2+)-chelate adsorbent and their ability to interact with full-length genomic TMV RNA was tested. Here we report that binding of viral RNA to hexahistidine fusion movement proteins results in the formation of stable ribonucleoprotein complexes.

Adsorption↗
PubMed1985

Ultrasonic absorption in tobacco mosaic virus and its protein aggregates.

The structural fluctuations specific to self-assembled biological systems have been investigated further with ultrasonic techniques by using two strains of tobacco mosaic virus (TMV), as well as the helical aggregate of the common strain protein and subassemblies of it. We confirmed our earlier conclusion that protein assemblies exhibit specific structural fluctuations detected in ultrasonic experiments. As in spherical viruses, the fluctuations exhibited by the protein aggregates having a quaternary structure similar to that of the virion were modified in the virus by interaction with the RNA strand. It is unlikely that the origin for the observed effect is due either to: (1) the difference in local mobility of the segment 89 to 113 of the polypeptide chain in TMV and in the helical aggregate on the one hand, and in smaller aggregates, on the other hand; or (2) a local fluctuation associated with proton transfer reactions or ion-pair interactions. The most remarkable feature in the TMV system is the fact that the two-ring disk showed no excess of ultrasonic absorption with respect to the A-protein oligomer, while a large increase of ultrasonic absorption was observed in the rod-like aggregate that had undergone the disk-helix transition.

Hydrogen-Ion Concentration↗
PubMed1986

Distribution of mass within native thick filaments of vertebrate skeletal muscle.

The distribution of mass within the vertebrate skeletal thick filament has been determined by scanning transmission electron microscopy. Thick and thin filaments from fresh rabbit muscle were mixed with tobacco mosaic virus (TMV), fixed with formaldehyde, dried onto thin carbon films and viewed in a computer-linked microscope. Electron scattering data from both TMV and thick filaments were analysed with reference to the long axis of the particles so that the distribution of mass within the particles could be determined. While TMV appeared to be a uniform rod at the resolution employed (4.3 nm), the thick filament was clearly differentiated along its length. M-line remnants at the centre of the filament were flanked by regions of low mass per unit length, corresponding to the bare zone of the filament, and then by the more massive cross-bridge regions. The mass per unit length was approximately constant through most of the cross-bridge zone and declined at the filament tips, in a manner consistent with a constant number of myosin molecules per 14.3 nm interval (crown) throughout the cross-bridge zone. Fourier analysis of the data failed to detect the expected 43 nm periodicity of C-protein. The total mass of the thick filament was 184 Mdalton (s.e.m., 1.6 X 10(6); n = 70). The mass of adhering M-line proteins was highly variable but, on average, was about 4 Mdalton. The total mass of the filament and the mass distribution in the cross-bridge zone are consistent with three myosin molecules per crown.

Actin Cytoskeleton↗
PubMed1987

Attenuated strains of tobacco mosaic virus. Reduced synthesis of a viral protein with a cell-to-cell movement function.

Attenuated strains of tobacco mosaic virus (TMV) have been used to protect crops against virulent strains. The synthesis of viral proteins and RNAs was investigated in protoplasts that had been infected separately with three tomato strains of TMV, virulent type L, and attenuated strains L11 and L11A. It was revealed that the mutations, which are responsible for the viral attenuation and have been mapped in the p126 (p184) gene, caused a reduction of the synthesis of the viral-coded p30 protein with a cell-to-cell movement function and its mRNA, but it had no significant effect on the synthesis of other viral proteins and RNAs in virus-infected protoplasts. Thus, it was shown that the attenuated strains can multiply as efficiently as the virulent strain in initially inoculated cells, but they can not spread efficiently outside the infected cells. In addition, it is suggested that a non-structural protein, p126 or p184, of TMV is involved in the synthesis of viral subgenomic p30 mRNA.

Mutation↗
PubMed1987

Purified scrapie prions resist inactivation by procedures that hydrolyze, modify, or shear nucleic acids.

Prions were purified from scrapie-infected hamster brains and incubated for 24 hr at 65 degrees with 2 mM Zn2+ or 5 mM Mg2+; no loss of infectivity was observed. Bacteriophage M13, tobacco mosaic virus (TMV), potato virus X, and potato spindle tuber viroid were all inactivated by divalent metal ions under these conditions. Prions also resisted inactivation by prolonged digestions with DNase I, RNases A and T1, and micrococcal nuclease. Prions were resistant to psoralen photoadduct formation using high concentrations of psoralens; in contrast, M13 bacteriophage was inactivated by low concentrations of all these psoralens. Hydroxylamine failed to inactivate prions even after lengthy exposures to concentrations as high as 1 M, while TMV and M13 were both inactivated. Sonication of prions failed to decrease infectivity even though rod-shaped aggregates were disrupted while both M13 and TMV lost infectivity.

Animals↗
PubMed1988

Hybrid brome mosaic virus RNAs express and are packaged in tobacco mosaic virus coat protein in vivo.

Brome mosaic virus (BMV) is an icosahedral virus with a tripartite RNA genome which infects monocotyledonous plants, while the cowpea or legume strain of tobacco mosaic virus (CcTMV) is a rod-shaped virus with a single component RNA genome which infects dicotyledonous plants. To examine the potential for exchanging entire genes between RNA viruses, biologically active cDNA clones were used to replace the natural coat gene of BMV RNA3 with the coat gene and encapsidation origin of CcTMV. In protoplasts coinoculated with BMV RNAs 1 and 2, the resulting hybrid RNA3 was replicated by BMV trans-acting factors but was packaged in TMV coat protein to give rod-shaped particles rather than the usual BMV icosahedra. When the CcTMV encapsidation origin was suitably inserted in derivatives of BMV RNAs 1 and 2, these RNAs were also packaged in a ribonuclease-resistant form in protoplasts coinoculated with the hybrid RNA3 expressing TMV rather than BMV coat protein. Thus, despite the markedly divergent nature of BMV and TMV, replicating hybrids bearing characters derived from both parent viruses were produced. Such hybrid viruses could be of considerable value for studying specific steps in infection and for assigning functions to particular virus genes.

Capsid↗
PubMed1988

Subcellular localization of tobacco mosaic virus minus strand RNA in infected protoplasts.

Radioactive RNA probes were prepared which specifically hybridize with sequences complementary to 5' and 3' regions of tobacco mosaic virus (TMV) RNA. These probes were used in Northern hybridization to locate TMV-RNA minus strands in the subcellular fractions of infected tobacco protoplasts. When the protoplasts were lysed with Triton X-100, full-length minus strands were present in the cytoplasmic but not in the nuclear fraction. With mechanically broken protoplasts, the crude nuclear fraction (250 g pellet) contained small amount of minus strands which appeared to derive from unbroken protoplasts, but most of minus strands were recovered in a fraction sedimented between 250 and 2500 g, little if any being found in lighter fractions. The results indicate that TMV-RNA replicates in association with an extranuclear structure.

Blotting, Northern↗
PubMed1988

Sequence and structure at the genome 3' end of the U2-strain of tobacco mosaic virus, a histidine-accepting tobamovirus.

The primary sequence of the 3' noncoding region of U2-TMV RNA was determined. A structural model was proposed based on chemical and enzymatic structure mapping as well as on analyses of nuclease protection by aminoacyl-tRNA-synthetase. The model agrees with those proposed for TMV "vulgare" RNA and confirms their general validity for the tobamoviruses. The RNA appears to have a tRNA-like, L-shaped structure at the 3' terminus, linked to a quasi-continuous double-helical stalk, with five pseudoknots involved in the formation of the whole structure. However, the structure of U2-TMV RNA is less stringently conserved than the 3' termini of "vulgare" and other histidine-accepting tobamoviruses. This difference is reflected in the kinetics of aminoacylation of the RNA.

Base Sequence↗
PubMed1989

Nucleotide sequence and translation of satellite tobacco mosaic virus RNA.

Satellite tobacco mosaic virus (STMV) is a plant virus with a 17-nm icosahedral particle encapsidating a 0.3 X 10(6) Mr ssRNA genome that depends on tobamoviruses for its replication. The complete nucleotide sequence of STMV RNA deduced in the experiments described here was 1059 nucleotides in length. The efficiency of labeling viral RNA with [gamma-32P]ATP using T4 polynucleotide kinase was not affected by treatment with tobacco acid pyrophosphatase and/or bacterial alkaline phosphatase, indicating that the majority of the 5' termini of encapsidated STMV RNAs were not phosphorylated. The 240 3'-terminal nucleotides of STMV RNA and either tobacco mosaic virus (TMV) U1 RNA or TMV U2/U5 RNA had greater than 65% overall sequence similarity, with two nearly identical regions of 40 and 50 bases, respectively. There were no other regions of sequence relatedness to TMV RNA. The 19 5'-terminal nucleotides of STMV RNA had greater than 65% sequence similarity with the 16 5'-terminal nucleotides of brome mosaic virus (RNA 3 and 50% sequence similarity with the 12 5'-terminal nucleotides of the Q strain of cucumber mosaic virus RNA 3. The first open reading frame (ORF) beginning at base 53 encoded a 6800 Mr protein that corresponded in size to a major in vitro translation product directed by STMV RNA. A second ORF, beginning at nucleotide 163, had the capacity to code for a protein that corresponded in size (17,500 Mr) to the other major in vitro translation product. The first 12 codons of this ORF corresponded to the sequence of the N-terminal amino acids of the capsid protein. Western-blot analysis of the in vitro translation products revealed that the 17,500 Mr protein had the same electrophoretic mobility as the authentic capsid protein; it was also antigenically related to the capsid protein, but the 6800 Mr protein was not. Time course analysis of in vitro translation demonstrated that the 6800 Mr protein was synthesized at the same time as the capsid protein and did not arise by the proteolytic cleavage of a larger precursor polypeptide. These results suggest that the genome of STMV functioned as a polycistronic messenger RNA. It has not been determined if the 6800 Mr protein is synthesized in vivo. STMV RNA had untranslated regions of 52 and 418 nucleotides at its 5' and 3' termini, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Changing the start codon context of the 30K gene of tobacco mosaic virus from "weak" to "strong" does not increase expression.

The translation initiation region of the 30K gene of tobacco mosaic virus (TMV) was modified by in vitro mutagenesis to create more optimal start codon contexts. A complicating factor was that modifications in this region also altered the 3' terminus of the 183K ORF that overlaps the 30K ORF. An insertion of GACUCGA between nucleotides 4901 and 4902 resulted in a purine (G) in position -3 relative to the AUG creating a "stronger" start codon context, but this also changed the last four amino acids of the 183K protein. This mutant was infectious, replicated efficiently, but produced reduced amounts of 30K protein. Despite the reduced amount of movement protein, this mutant spread effectively from cell to cell and had a phenotype indistinguishable from that of wild-type virus. A more conservative mutation inserted GAC between TMV nucleotides 4901 and 4902 resulting in a "strong" start codon context (ACGAUGG) and modification of the 183K protein only by insertion of an aspartic acid adjacent to a native aspartic acid. This modification did not enhance the production of 30K protein. These data demonstrate consensus sequences that are optimal for other eukaryotic systems did not cause increased expression of the 30K gene in vivo. The modified sequences of both mutants were stably maintained during relatively long periods of replication. Even though each mutant replicated efficiently, when mixed with wild-type TMV, neither mutant effectively competed with the wild-type virus. Another mutant which removed the native 30K AUG to determine whether subsequent internal start codons with "stronger" contexts would function in its absence was constructed. However, this mutant and a mutant that fused the 183K reading frame to the 30K reading frame did not replicate and move in intact plants.

Base Sequence↗