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PubMed2003

Identification of a region of the tobacco mosaic virus 126- and 183-kilodalton replication proteins which binds specifically to the viral 3'-terminal tRNA-like structure.

UV irradiation of a mixture of an isolated tobacco mosaic virus (TMV; tomato strain L [TMV-L]) RNA-dependent RNA polymerase complex and the TMV-L RNA 3'-terminal region (3'-TR) resulted in cross-linking of the TMV-L 126-kDa replication protein to the TMV-L 3'-TR. Using both Escherichia coli-expressed proteins corresponding to parts of the 126-kDa protein and mutants of the 3'-TR, the interacting sites were located to a 110-amino-acid region just downstream of the core methyltransferase domain in the protein and a region comprising the central core C and domain D2 in the 3'-TR. Mutation to alanine of a tyrosine residue at position 409 or a tyrosine residue at position 416 in the protein binding region abolished cross-linking to the 3'-TR, and corresponding mutations introduced into TMV-L RNA abolished its ability to replicate in tomato protoplasts, with no detectable production of either plus- or minus-strand RNA. The results are compatible with a model for initiation of TMV-L minus-strand RNA synthesis in which an internal region of the TMV-L 126-kDa protein first binds to the central core C and domain D2 region of the TMV-L 3'-TR and is then followed by binding of the 183-kDa protein to this complex and positioning of the catalytically active site of the polymerase domain close to the 3'-terminal CCCA initiation site.

Amino Acid Sequence↗

PubMed1996

Endothelin-1 depresses tracheal mucus velocity in ovine airways via ET-A receptors.

Endothelin-1 (ET-1) is a potent constrictor of bronchial smooth muscle, but there is limited information on its actions on the airway mucociliary clearance in vivo. The purpose of this study was to determine (1) the effect of aerosolized ET-1 on tracheal mucus velocity (TMV), a marker of mucociliary clearance, in sheep and (2) if the ET-1-induced effects were mediated by ET-A or ET-B receptors. To measure TMV, radiopaque teflon particles were insufflated into six intubated, spontaneously breathing, adult sheep, and the velocity at which these particles traveled up the trachea was measured using a previously reported roentgenographic technique. After baseline TMV measurements, 50 breaths of either ET-1 (10(-7) M) or vehicle (phosphate-buffered saline) were aerosolized into the airways. TMV measurements were then obtained over a 2-h period. After exposure to ET-1, mean TMV decreased significantly as compared with vehicle, the effects being most marked within 30 min after administration (54%, p < 0.05). On subsequent days, animals were pretreated with an aerosolized ET-A receptor antagonist (BQ-123) or an ET-B receptor antagonist (BQ-788) before exposure to ET-1. When ET-1 was given after BQ-123, no significant drop in TMV was noted. In contrast, pretreatment with BQ-788 exhibited no protective effect on the decrease in TMV. The ET-1 effects were not influenced by pretreatment with either the cyclo-oxygenase inhibitor indomethacin or the leukotriene receptor antagonist MK-571, indicating that ET-1-induced depression in TMV does not involve the activation of prostanoids or peptide leukotrienes. Thus, exogenous ET-1 reduces TMV, an in vivo effect that is mediated through stimulation of ET-A receptors.

Aerosols↗

PubMed1978

Mucociliary transport in allergic patients with antigen-induced bronchospasm.

Tracheal mucous velocity (TMV) and respiratory mechanics were measured in asymptomatic asthmatic patients with ragweed hypersensitivity before and after inhalation of specific antigen, and with or without cromolyn sodium pretreatment. TMV was measured radiographically, and the airway response to bronchial provocation was monitored by measurements of forced expiratory volume in one sec and specific airway conductance. TMV was significantly less (6.3 +/- 2.3 mm per min, mean +/- SD) in the 6 asymptomatic asthmatic patients than in 7 normal subjects (11.6 +/- 3.6 mm per min, mean +/- SD). In the asthmatic patients, mean TMV diminished to 72 per cent of baseline immediately after bronchial provocation when specific airway conductance was decreased to 65 per cent of baseline or less, with a further decrease in TMV to 47 per cent of baseline after one hour, at which time respiratory mechanics had returned to baseline values. Pretreatment with cromolyn sodium prevented the decrease in TMV after bronchial provocation. We concluded that in asymptomatic patients with allergic asthma, (1) baseline TMV is impaired, (2) inhalation of specific antigen causes a marked decrease in TMV independent of the degree of bronchospasm, and (3) the decrease in TMV may be related to the release of chemical mediators.

Adolescent↗

PubMed2002

A hot pepper cDNA encoding ascorbate peroxidase is induced during the incompatible interaction with virus and bacteria.

Capsicum annuum L. is infected by a number of viruses, including the tobacco mosaic virus (TMV). To study the defense-related genes that are induced by TMV in hot peppers, the pepper plant, which is susceptible to P1.2 but resistant to the P0 pathotype of TMV, was inoculated with TMV-P0. Differential screening isolated the genes that were specifically up- or down-regulated during the hypersensitive response (HR). The CaAPX1 cDNA clone that putatively encodes a polypeptide of cytosolic ascorbate peroxidase was selected as an up-regulated gene. It was isolated for further study. The full-length cDNA for CaAPX1, which is 972 bp long, contained the open-reading frame of 250-amino acid residues. A genomic Southern blot analysis showed that there were only limited copies of the CaAPX1 gene in the hot pepper genome. In hot pepper cv. Bugang, which is resistant to TMV-P0 and susceptible to TMV-P1.2, the CaAPX1 gene transcript was accumulated by TMV-P0, but not by TMV-P1.2 inoculation. CaAPX1 transcripts began to accumulate 24 h post-inoculation of TMV-P0, and increased gradually until 96 h. To investigate whether each transcript is induced by other stimuli, the plants were treated with various chemicals and wounding. A striking induction of the CaAPX1 transcript was observed at 2 h. It subsided 12 h after salicylic acid (SA), ethephon, and methyl jasmonate (MeJA) treatments. The response of the gene upon other pathogen infection was also examined by a bacterial pathogen (Xanthomonas campestris pv. vesicatoria race 3) inoculation. The CaAPX1 gene was induced in a hot pepper (C. annuum cv. ECW 20R) that was resistant to this bacterial pathogen, but not in a susceptible hot pepper (C. annuum cv. ECW). These results suggest the possible role(s) for the CaAPX1 gene in plant defense against viral and bacterial pathogen.

Amino Acid Sequence↗

PubMed1994

Immunosorbent electron microscopic studies on a tobamovirus causing brinjal necrotic mosaic.

Serological relationships of brinjal necrotic mosaic virus (BNMV), a strain of tobacco mosaic virus (TMV) causing necrotic mosaic disease of brinjal in India to other TMV strains was investigated by immunosorbent electron microscopy (ISEM). The intensity of trapping and decoration revealed a close relationship of BNMV to TMV-D, TMV-U1 and TMV-WU1 strains, and a distant relationship to TMV-A1 and TMV-P11 strains. There was a negligible relationship to TMV-P14, tomato mosaic virus (ToMV) and cucumber green mottle mosaic virus (CGMMV). Therefore, BNMV is proposed to be distinct from the previously reported TMV-A1 strain of brinjal.

India↗

PubMed1999

Display of epitopes on the surface of tobacco mosaic virus: impact of charge and isoelectric point of the epitope on virus-host interactions.

The biophysical properties of the tobacco mosaic tobamovirus (TMV) coat protein (CP) make it possible to display foreign peptides on the surface of TMV. The immunogenic epitopes G5-24 from the rabies virus (RV) glycoprotein, and 5B19 from murine hepatitis virus (MHV) S-glycoprotein were successfully displayed on the surface of TMV, and viruses accumulated to high levels in infected leaves of Nicotiana tabacum Xanthi-nn. The peptide RB19, which contains an arginine residue plus the 5B19 epitope fused to the CP (TMV-RB19), resulted in the induction of necrotic local lesions on inoculated leaves of N. tabacum Xanthi-nn and cell death of infected BY2 protoplasts. RNA dot blot assays confirmed that expression of the acidic and basic pathogenesis-related PR2 genes were induced in infected Xanthi-nn leaf tissue. TMV that carried epitope 31D from the RV nucleoprotein did not accumulate in inoculated tobacco leaves. Analysis of hybrid CPs predicted that the isoelectric points (pI):charge value was 5.31:-2 for wild-type CP, 5.64:-1 for CP-RB19, and 9.14:+2 for CP-31D. When acidic amino acids were inserted in CP-RB19 and CP-31D to bring their pI:charge to near that of wild-type CP, the resulting viruses TMV-RB19E and TMV-4D:31D infected N. tabacum Xanthi-nn plants and BY2 protoplasts without causing cell death. These data show the importance of the pI of the epitope and its effects on the hybrid CP pI:charge value for successful epitope display as well as the lack of tolerance to positively charged epitopes on the surface of TMV.

Amino Acid Sequence↗

PubMed1996

Mapping nucleotides in the 126-kDa protein gene that control the differential symptoms induced by two strains of tobacco mosaic virus.

The differential symptom determinants of the Holmes' masked (M) and U1 strains of tobacco mosaic virus previously were mapped to the 5'-coterminal open reading frame (ORF) encoding the 126-kDa protein and the N-terminal two-thirds of the 183-kDa protein. Both proteins influence viral RNA accumulation, but the function of, and impact on, symptom formation by large domains within the 126-kDa gene, which are not conserved with sequences in analogous ORFs from other related viruses, are unknown. In the current study, cDNA clones representing each strain (i.e., MIC-TMV and U1-TMV) were mutated in these nonconserved domains to further define the nucleotides responsible for mosaic symptom induction on Nicotiana tabacum. Progeny virus of a mutant containing only eight nucleotide substitutions from the MIC-TMV sequence to the U1-TMV sequence within the 126-kDa protein ORF of MIC-TMV induced U1-TMV-like symptoms. Single or multiple substitutions among these eight nucleotides further defined residues critical for symptom modulation. Complementary substitutions in the MIC-TMV and U1-TMV sequences did not always yield progeny virus that induced complementary visual symptoms. Progeny of some mutants contained second-site spontaneous mutations at specific positions shown to influence symptom phenotype. For a subset of the stable site-directed mutants, there was no correlation between severity of systemic symptoms and chlorotic lesion size or virus accumulation in these chlorotic lesions on inoculated leaves.

Base Sequence↗

PubMed2000

Tobacco mosaic virus replicase-mediated cross-protection: contributions of RNA and protein-derived mechanisms.

Specific sequences of the tobacco mosaic virus (TMV) RNA-dependent RNA-polymerase (RdRp) gene were investigated for their ability to confer cross-protection. Nine overlapping segments ranging from 713 to 1070 nucleotides in length and covering the methyltransferase, helicase, and polymerase (POL) domains of the TMV RdRp open reading frame were systemically expressed in Nicotiana benthamiana using a potato X virus (PVX) vector [Chapman, S., Kavanagh, T., and Baulcombe, D. C. (1992). Plant J., 1, 549-557]. PVX-infected plants were subsequently challenge inoculated with 10 microg of wild-type TMV and monitored for TMV accumulation. Mock inoculated plants and plants preinfected with the unmodified PVX vector rapidly accumulated high levels of challenge virus. In contrast, plants preinfected with PVX vectors expressing segments of the TMV RdRp open reading frame displayed either high or low levels of protection. High protection levels were observed for PVX constructs expressing segments of the TMV POL domain, whereas low protection levels were observed for PVX constructs expressing segments covering the methyltransferase and helicase domains. Frameshift mutations that blocked protein expression from RdRp segments disrupted only the high levels of protection derived from POL segments and not the low levels derived from the other segments. However, all RdRp segments conferred similarly high levels of protection against a TMV construct with restricted local movement. Thus both RNA and protein sequences in conjunction with the speed of the infecting challenge virus can affect the protection derived from the TMV RdRp gene.

Frameshift Mutation↗

PubMed2002

Silencing of a gene encoding a protein component of the oxygen-evolving complex of photosystem II enhances virus replication in plants.

It has been suggested that, in addition to viral proteins, host proteins are involved in RNA virus replication. In this study the RNA helicase domain of the Tobacco mosaic virus (TMV) replicase proteins was used as bait in the yeast two-hybrid system to identify tobacco proteins with a putative role in TMV replication. Two host proteins were characterized. One protein (designated #3) belongs to a protein family of ATPases associated with various activities (AAA), while the second host protein (designated #13) is the 33K subunit of the oxygen-evolving complex of photosystem II. Using Tobacco rattle virus vectors, genes #3 and #13 were silenced in Nicotiana benthamiana, after which the plants were challenged by TMV infection. Silencing of gene #13 resulted in a 10-fold increase of TMV accumulation, whereas silencing of gene #3 caused a twofold reduction of TMV accumulation. Additionally, silencing of genes #3 and #13 decreased and increased, respectively, the accumulation of two other viruses. Similar to silencing of gene #13, inhibition of photosystem II by application of an herbicide increased TMV accumulation several fold. Infection of N. benthamiana with TMV resulted in a decrease of #13 mRNA levels. Silencing of gene #13 may reflect a novel strategy of TMV to suppress basal host defense mechanisms. The two-hybrid screenings did not identify tobacco proteins involved in helicase domain-induced N-mediated resistance.

Adenosine Triphosphatases↗

PubMed1991

Rat hind-paw swelling effect of an edema-producing protein isolated from Trimeresurus mucrosquamatus snake venom.

TMV F-IV, isolated from the venom of Trimeresurus mucrosquamatus (TMV), caused rat hind-paw edema in a dose-dependent manner. The maximum hind-paw swelling was reached at 1.5-2 h after subplantar injection of TMV F-IV. The edematous response caused by TMV F-IV was suppressed by the s.c. pretreatment with diphenhydramine, methysergide, acetylsalicylic acid or dexamethasone, and by the subplantar co-injection with FPL 55712, a SRS-A antagonist, and BN 52021 or L 652731, both PAF antagonists. Polymorphonuclear (PMN) leukocyte infiltration appeared within 1 h and gradually increased in the rat paw 3-6 h after edema induction. Compound 48/80 or methotrexate pretreatment also inhibited paw edema caused by TMV F-IV. In isolated mast cells, TMV F-IV increased the formation of PGE2 and LTB4 and caused a dose-dependent release of histamine and beta-glucuronidase. Since there are no significant differences in paw edema and mast cell degranulation responses between TMV F-IV and its DFP-modified analogue, the esterase activity may not be necessary in these models. These results indicate that mast cells. PMN leukocytes and some inflammatory mediators such as histamine, serotonin, arachidonate metabolites and PAF are involved in TMV F-IV induced paw edema.

Animals↗

PubMed1989

Assembly of hybrid RNAs with tobacco mosaic virus coat protein. Evidence for incorporation of disks in 5'-elongation along the major RNA tail.

We have shown that during the reassembly of tobacco mosaic virus (TMV) RNA, with the coat protein supplied as a "disk preparation", the lengths of RNA protected from nuclease are "quantized" with steps which correspond to incorporation of the subunits from either a single or, more commonly, both rings of a disk. This interpretation has been challenged and it was suggested that the pattern was due to special, though unspecified features of the sequence of TMV RNA. To test whether the specific sequence of TMV RNA is important during the elongation, rather than just during nucleation, we have now followed growth of particles containing hybrid RNAs, with the TMV RNA origin of assembly but otherwise non-TMV sequences. We have prepared in vitro RNA transcripts containing heterologous RNA 5' to the origin of assembly sequence from TMV RNA, i.e. with a heterologous RNA tail in place of the natural major 5'-tail and no minor tail, and used these for assembly experiments. In each case we observe a banding pattern very similar to that which we had found with native TMV RNA and with a dominant quantum step of just over 100 bases, and sometimes also a step of 50 bases, strongly suggesting that this is not due to any feature of the TMV RNA. This same repeat is also visible even with a heterologous RNA chosen because it had a sequence repeat of 135 or 136 bases, confirming that the quantization is due to a feature of the elongation reaction and in no way to the RNA sequence being encapsidated. We have also followed elongation with the origin of assembly located 5' to the heterologous RNA. This leads to a slower elongation along this 3'-tail, after the initial rapid encapsidation of the origin RNA, which lacks any quantization of length protected. These results are fully compatible with the hypothesis we had advanced earlier, that the major growth along the 5'-tail is from performed aggregates ("disks") while the minor growth along the 3'-tail is from subunits in the "A-protein" adding singly or a few at a time.

Base Sequence↗

PubMed1991

Transfer of the movement protein gene between two tobamoviruses: influence on local lesion development.

The effects of transfer of the movement gene between the tobamoviruses tobacco mosaic virus (TMV) and tobacco mild green mosaic virus (TMGMV) were studied. The movement protein (MP) gene of TMGMV was cloned into an infectious cDNA of TMV to build the recombinant virus V23. V23, like TMV and TMGMV, caused systemic infection in Nicotiana tabacum Xanthi. In N. sylvestris V23 and TMV spread systemically although TMGMV produces necrotic local lesions on this host. V23 and TMV cause systemic infection on tomato plants while TMGMV does not infect tomato. In Xanthi nc plants, V23 produced necrotic local lesions similar in size to those produced by TMGMV. On the other hand in transgenic Xanthi nc tobacco plants that express a gene encoding the MP of TMV the necrotic lesions produced by V23 and TMGMV were similar in size to those produced by TMV. These results indicate that the size of necrotic lesions produced by TMGMV and TMV on Xanthi nc plants is influenced by the MP gene.

Amino Acid Sequence↗

PubMed1995

Solution X-ray scattering study of reconstitution process of tobacco mosaic virus particle using low-temperature quenching.

The reconstitution process of tobacco mosaic virus (TMV) was investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. TMV assembly in an aqueous solution is completely stopped below 5 degrees C. The TMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5 degrees C on a series of solutions prepared by low-temperature quenching after incubation either at 15, 20 or 25 degrees C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. In order to account the time course of SAXS profiles in terms of the elongation of TMV assembly, a model calculation was performed to simulate the Guinier plot, the Kratky plot and the distance distribution function by applying Glatter's multibody method using models that were constituted of the spheres representing a column of piled two-layer disks of TMV-protein. The three simulated functions thus obtained support the conclusion derived from the three functions calculated from the experimental results that the incubation of the RNA and protein of TMV began to reconstitute TMV instantly after mixing, proceeded steeply to a long rod, and then extended asymptotic to the full length of the TMV particle. This process is in good agreement with that obtained from electron microscopic studies.

Journal Article↗

PubMed1992

Radial mass density functions of vitrified helical specimens determined by scanning transmission electron microscopy: their potential use as substitutes for equatorial data.

Using STEM dark field images, we have determined linear mass densities and radial density profiles of vitrified helical particles. The samples studied are: TMV, RNA-free helical polymers of TMV coat protein (TMV-P), Salmonella typhimurium bacterial flagellar filaments and Escherichia coli pili. The difference between the profiles obtained for TMV and TMV-P shows a maximum at a radius of about 4 nm, corresponding to the RNA in TMV. Of the peaks that are resolved in X-ray diffraction analysis we can resolve the ones for TMV at radii of approximately 4.2 and approximately 6.7 nm and a shoulder at approximately 7.8 nm. Density peaks in bacterial flagellar filaments appear at radii of approximately 4.2, approximately 6.5, approximately 8.5, and approximately 10.5 nm. Accurate mass data can be obtained if the filaments are embedded in ice layers of uniform thickness; their diameters need to be similar to that of the mass standard (TMV) when these data are measured in a comparative manner. Ice layers are often not uniform, and thickness variations are well revealed in STEM dark field. The signal-to-noise ratio and contrast for the transverse projections are lower than those measured for freeze-dried specimens: half an order and one order of magnitude, respectively. The thinnest uniformly thick ice layer still containing a single layer of particles is approximately 10-15 nm thicker than the particles. Radial mass density functions that are directly determined in STEM may have a potential use as substitutes for the unreliable equatorial data in helical reconstructions of TEM bright field images of vitrified specimens.

Capsid↗

PubMed1980

Studies on the mechanism of assembly of tobacco mosaic virus.

Sedimentation and proton binding studies on the endothermic self-association of tobacco mosaic virus (TMV) protein indicate that the so-called "20S" sedimenting protein is an interaction system involving at least the 34-subunit two-turn yield cylindrical disk aggregate and the 49-subunit three-turn helical rod. The pH dependence of this overall equilibrium suggests that disk formation is proton-linked through the binding of protons to the two-turn helix which is not present as significant concentrations near pH 7. There is a temperature-induced intramolecular conformation change in the protein leading to a difference spectrum which is complete in 5 x 10(-6) s at pH 7 and 20 degrees C and is dominated at 300 nm by tryptophan residues. Kinetics measurements of protein polymerization, from 10(-6) to 10(3) s, reveal three relaxation processes at pH 7.0, 20 degrees C, 0.10 M ionic strength K (H) PO4. The fastest relaxation time is a few milliseconds and represents reactions within the 4S protein distribution. The second fastest relaxation is 50-100 x 10(-3) s and represents elementary polymerization steps involved in the formation of the approximately 20 S protein. Analysis of the slowest relaxation, approximately 5 x 10(4) s, suggests that this very slow formation of approximately 20 S protein may be dominated by some first order process in the overall dissociation of approximately 20S protein. Sedimentation measurements of the rate of TMV reconstitution, under the same conditions, show by direct measurements of 4S and approximately 20S incorporation at various 4S to approximately 20S weight ratios that the relative rate of approximately 20S incorporation decreases almost linearly, from 0 to 50% 4S. There appears to be one or more regions of TMV-RNA, approximately 1-1.5 kilobases long, which incorporates approximately 20S protein exclusively. Solutions of approximately 95-100% approximately 20S protein have been prepared for the first time and used for reconstitution with RNA. Such protein solutions yield full size TMV, but at a slower rate than if 4S protein is added. Thus the elongation reaction in TMV assembly, following nucleation with approximately 20S protein, is not exclusively dependent upon the presence of either 4S or approximately 20S protein aggregates. The initial, maximum, rate of reconstitution increases about threefold when the protein composition is changed from 5% to 30% 4S protein, at constant total protein concentration at pH 7.0, 20 degrees C in 0.10 M ionic strength K (H)PO4. The probable binding frame at the internal assembly nucleation site of TMV-RNA has been determined by measuring the association constants for the binding of various trinucleoside diphosphates to helical TMV protein rods. The -CAG-AAG-AAG-sequence at the nucleation site is capable of providing at least 10-14 kcal/mol of sites of binding free energy for the nucleation event in TMV self-assembly.

Base Sequence↗

PubMed1975

The specificity of different classes of ethylating agents toward various sites in RNA.

The alkyl products of neutral in vitro ethylation of TMV-RNA by [14C]diethyl sulfate, [14C]ethyl methanesulfonate, and [14C]ethylnitrosourea have been determined and found to differ significantly depending on the ethylating agent. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of TMV-RNA in the following order: 7-ethylguanine greater than 1-ethyladenine, 3-ethylcytidine greater than 7-ethyladenine, 3-ethyladenine, O6-ethylguanosine, 3-ethylguanine. Ethyl methanesulfonate was more specific for the 7 position of guanine, and other derivatives were found in lesser amounts than with diethyl sulfate. Neither reagent caused the formation of detectable amounts (smaller than 0.26 percent) of 1-ethylguanine, 1,7-diethylguanine, N2-ethylguanine, N6-ethyladenine, N4-ethylcytidine, or 3-ethyluridine. Identified ethyl bases account for over 85% of the total radioactivity of [14C]ethyl methanesulfonate and [14C]diethyl sulfate treated TMV-RNA. Phosphate alkylation accounts for about 13 and 1%, respectively, In contrast, [14C]ethylnitrosourea-treated TMV-RNA, while reacting to a similar extent (15-70 ethyl groups/6400 nucleotides), is found to cause considerably more phosphate alkylation. Upon either U4A RNase or acid hydrolysis up to 60% of the radioactivity is found as volatile ethyl groupw in the form of [14C]ethanol, and a further 15% appears to be primarily ethyl phosphate and nucleosides with ethylated phosphate. Of the remaining radioactivity, half is found as O6-ethylguanosine, the major identified ethyl nucleoside. Other ethyl bases found in ethylnitrosourea-treated TMV-RNA are 7-ethylguanine greater than 1-ethyladenine, 3-ethyladenine, 7-ethyladenine, 3-ethylcytidine, and 3-ethylguanine. It appears that ethylnitrosourea preferentially alkylates oxygens, and that formation of phosphotriesters is by far the predominant chemical event. Since the number of ethyl groups introduced into TMV-RNA by ethylnitrosourea is similar to the number of lethal events, one may conclude that phosphate alkylation leads to loss of infectivity. None of the three ethylating agents studied are strongly mutagenic on TMV-RNA or TMV. The role of phosphate alkylation in regard to in vivo mutagenesis and oncogenesis remains to be established. At present it appears possible that the extent of this reaction may correlate better with the oncogenic effectiveness of different ethylating agents, than the extent of any base reaction. Unfractionated HeLa cell RNA is ethylated primarily in acid labile manner even by diethyl sulfate and ethyl methanesulfonate, a fact that is attributed to its high content of low molecular weight trna rich in terminal phosphates which alkylate readily.

Alkylating Agents↗

PubMed1994

Expression of tobacco mosaic virus coat protein and assembly of pseudovirus particles in Escherichia coli.

The bidirectional self-assembly of tobacco mosaic virus (TMV, common or U1 strain) has been studied extensively in vitro. Foreign single-stranded RNA molecules containing the TMV origin-of-assembly sequence (OAS, 75-432 nt in length) are also packaged by TMV coat protein (CP) in vitro to form helical pseudovirus particles. To study virus assembly in vivo requires an easily manipulated model system, independent of replication in plants. The TMV assembly machinery also provides a convenient means to protect and recover chimeric gene transcripts of almost any length or sequence for a variety of applications. Native TMV CP expressed in and purified from Escherichia coli formed nonhelical, stacked aggregates after dialysis into pH 5 buffer and was inactive for in vitro assembly with TMV RNA. U1 CP derivatives in which the second amino acid was changed from Ser to Ala or Pro, nonacetylated N termini found in two natural strains of the virus, failed to remediate these anomalous properties. However, in vivo coexpression of CP and single-stranded RNAs (up to approximately 2 kb) containing the TMV OAS gave high yields of helical pseudovirus particles of the predicted length (up to 7.4 +/- 1.4 micrograms/mg of total bacterial protein). If the OAS-containing RNA was first recruited into bacterial polyribosomes, elongation of pseudovirus assembly was blocked. In vivo, E. coli expression of a full-length cDNA clone of the TMV genome (6.4 kb) resulted in high, immunodetectable levels of CP and assembly of sufficient intact genomic RNA to initiate systemic infection of susceptible tobacco plants.

Base Sequence↗

PubMed1994

Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability.

Plasmodesmata are cytoplasmic bridges between plant cells thought to generally allow only the passage of small molecules and metabolites. However, large structures such as plant viruses also move from cell to cell via plasmodesmata. In tobacco mosaic virus (TMV) infection a viral movement protein (TMV-MP) mediates viral spread. Here, a microinjection assay is used to monitor the dynamics of TMV-MP function directly in wild-type plants. The results indicate that TMV-MP interacts with an endogenous plant pathway increasing plasmodesmal size exclusion limit to permit passage of 20-kDa dextrans. Furthermore, TMV-MP influences plasmodesmal size exclusion limit several cells distant from the injection site, indicating either that TMV-MP itself crosses plasmodesmata or that TMV-MP induces a diffusable signal capable of dilating microchannels of plasmodesmata. The region of TMV-MP responsible for increasing plasmodesmal size exclusion limit was mapped to the carboxyl-terminal part of the 268-amino acid residue protein between amino acid residues 126 and 224.

Biological Assay↗