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PubMed2000

[Complete nucleotide sequences and genome structure of two Chinese tobacco mosaic virus isolates deduced from full-length infectious cDNA clones].

The complete nucleotides of two Chinese tobacco mosaic virus (TMV) isolates, TMV-Cv (vulgare strain) and TMV-N14 (an attenuated virus originated from a tomato strain), were determined from their respective full-length infectious cDNA clones and compared with published TMV sequences. The genome structure of TMV-Cv contained 6395 nucleotides, in which four functional open reading frames (ORF), coding for replicase (126 kD/183 kD), movement protein (MP, 30 kD) and coat protein (CP, 17.6 kD) respectively, could be recognized. TMV-N14 contained 6384 nucleotides in its genome. In contrast to TMV-Cv, five functional ORFs encoding the replicase 98.5 kD/126 kD/183 kD, MP(27 kD) and CP(17.6 kD), respectively, were detected in the TMV-N14 genome. TMV-Cv is 99% homologous to a Korean TMV isolate belonging to the vulgare strain at the nucleotide level. TMV-N14 is 99% homologous to a highly virulent Japanese isolate TMV-L (tomato strain) at the nucleotide level. In TMV-N14, one opal nulation (UGA) occurred in the replicase gene and one ochre nutation (UAA) in the MP gene. The former mutation created a potential, additional ORF within the replicase gene, the latter reduced the size of the MP to 27 kD. In addition, there were also 13 amino acid substitutions in the replicase gene of TMV-N14 when compared to that of TMV-L. Collectively, these changes may have significant implications in the attenuation of the virulence of TMV-N14.

Amino Acid Sequence↗
PubMed1999

Historical overview of research on the tobacco mosaic virus genome: genome organization, infectivity and gene manipulation.

Early in the development of molecular biology, TMV RNA was widely used as a mRNA [corrected] that could be purified easily, and it contributed much to research on protein synthesis. Also, in the early stages of elucidation of the genetic code, artificially produced TMV mutants were widely used and provided the first proof that the genetic code was non-overlapping. In 1982, Goelet et al. determined the complete TMV RNA base sequence of 6395 nucleotides. The four genes (130K, 180K, 30K and coat protein) could then be mapped at precise locations in the TMV genome. Furthermore it had become clear, a little earlier, that genes located internally in the genome were expressed via subgenomic mRNAs. The initiation site for assembly of TMV particles was also determined. However, although TMV contributed so much at the beginning of the development of molecular biology, its influence was replaced by that of Escherichia coli and its phages in the next phase. As recombinant DNA technology developed in the 1980s, RNA virus research became more detached from the frontier of molecular biology. To recover from this setback, a gene-manipulation system was needed for RNA viruses. In 1986, two such systems were developed for TMV, using full-length cDNA clones, by Dawson's group and by Okada's group. Thus, reverse genetics could be used to elucidate the basic functions of all proteins encoded by the TMV genome. Identification of the function of the 30K protein was especially important because it was the first evidence that a plant virus possesses a cell-to-cell movement function. Many other plant viruses have since been found to encode comparable 'movement proteins'. TMV thus became the first plant virus for which structures and functions were known for all its genes. At the birth of molecular plant pathology, TMV became a leader again. TMV has also played pioneering roles in many other fields. TMV was the first virus for which the amino acid sequence of the coat protein was determined and first virus for which cotranslational disassembly was demonstrated both in vivo and in vitro. It was the first virus for which activation of a resistance gene in a host plant was related to the molecular specificity of a product of a viral gene. Also, in the field of plant biotechnology, TMV vectors are among the most promising. Thus, for the 100 years since Beijerinck's work, TMV research has consistently played a leading role in opening up new areas of study, not only in plant pathology, but also in virology, biochemistry, molecular biology, RNA genetics and biotechnology.

Capsid↗
PubMed1998

Coat protein interactions involved in tobacco mosaic tobamovirus cross-protection.

To investigate the molecular role of the tobacco mosaic tobamovirus (TMV) coat protein (CP) in conferring cross-protection, a potato X potexvirus (PVX) vector (S. Chapman, Plant J. 2, 549-557, 1992) was used to systemically express a set of TMV mutant CPs in Nicotiana benthamiana prior to challenge inoculation with TMV. PVX-expressed wild-type TMV CP delayed TMV accumulation for up to 2 weeks compared to unprotected plants or plants preinfected with the unmodified PVX vector. Similar delays in TMV accumulation were obtained using TMV CPs that were deficient in virion formation but competent to assemble into helical aggregates. In contrast, TMV CPs that were incapable of helical aggregation or unable to bind viral RNA did not delay the accumulation of TMV. Furthermore, TMV CPs with enhanced intersubunit interactions that favor helical aggregation produced significantly greater delays in the accumulation of challenge TMV than obtained from the wild-type CP. Thus the capabilities of TMV CP to interact with viral RNA and self-associate in a helical fashion appear to be essential to its ability to confer protection. Taken together, these findings support a model for CP-mediated resistance in which the protecting CP recoats the challenge virus RNA as it disassembles.

Capsid Proteins↗
PubMed2002

Characterization of mutant tobacco mosaic virus coat protein that interferes with virus cell-to-cell movement.

Expression of tobacco mosaic virus (TMV) coat protein (CP) in plants confers resistance to infection by TMV and related tobamoviruses. Certain mutants of the CP (CP(T42W)) provide much greater levels of resistance than wild-type (wt) CP. In the present work, infection induced by RNA transcripts of TMV clones that contain wt CP or mutant CP(T42W) fused to the green fluorescent protein (GFP) (TMV-CP:GFP, TMV-CP(T42W):GFP) and clones harboring TMV movement protein (MP):GFP were followed in nontransgenic and transgenic tobacco BY-2 protoplasts and Nicotiana tabaccum Xanthi-nn plants that express wt CP or CP(T42W). On nontransgenic and wt CP transgenic plants, TMV-CP:GFP produced expanding, highly fluorescent disk-shaped areas. On plants expressing CP(T42W), infection by TMV-CP:GFP or TMV-MP:GFP-CP produced infection sites of smaller size that were characterized by low fluorescence, reflecting reduced levels of virus spread and reduced accumulation of both CP:GFP and MP:GFP. TMV-CP(T42W):GFP failed to produce visible infection sites on nontransgenic plants, yet produced normal infection sites on MP-transgenic plants that produce MP. TMV infection of transgenic BY-CP(T42W) protoplasts resulted in very low levels of MP accumulation, whereas on BY-CP protoplasts (containing wt CP), infection produced higher levels of MP than in nontransgenic BY-2 cells. The results suggest that wt CP has a positive effect on the production of MP, whereas the CP(T42W) has a negative effect on MP accumulation and/or function. This effect results in very high levels of resistance to TMV infection in plants containing CP(T42W). This report shows that the CP of a plant virus regulates production of the MP, and that a mutant CP interferes with MP accumulation and cell-to-cell movement of infection.

Amino Acid Substitution↗
PubMed1998

Specific sequence changes in the 5'-terminal region of the genome of satellite tobacco mosaic virus are required for adaptation to tobacco mosaic virus.

The genome of satellite tobacco mosaic virus (STMV) adapted to tobacco mosaic virus (TMV), tomato mosaic virus or green tomato atypical mosaic virus consistently had two single base deletions at positions 1 and 61, corresponding to bases A and G, respectively, as compared to the type-strain genome which is naturally adapted to tobacco mild green mosaic virus (TMGMV). Transcript RNAs (STMV(TMV)) from clone pSTMV(TMV) which captured the deletions at positions 1 and 61 were infectious when co-inoculated to tobacco plants with either TMV or TMGMV at infection frequencies of > 90%. Two new STMV variants were created to investigate whether both deletions were essential for adaptation to TMV. These were STMV(TMGMV) deltaA1, which had the A at position 1 (A1) deleted, and STMV(TMGMV) deltaG61, which lacked G61. STMV(TMGMV) deltaA1 was infectious (75% frequency) in the presence of either TMV or TMGMV. Virion RNA of STMV(TMGMV) deltaA1 lost G61 after one infection cycle with TMV. This deletion did not occur in co-infections with TMGMV. STMV(TMGMV) deltaG61, like the clone STMV(TMGMV), was infectious (100% frequency) with TMGMV but TMV did not support this clone. When Nicotiana benthamiana protoplasts were transfected with STMV(TMGMV), STMV(TMGMV) deltaA1 or STMV(TMV), STMV replicated when TMGMV was the helper virus. STMV(TMV) and STMV(TMGMV) deltaA1 replicated in the presence of helper TMV, but STMV(TMGMV) did not, the same result as in whole plants. The deletion of A1 is thus essential for initial STMV adaptation to TMV and the eventual deletion of G61 is a predicted additional change.

Adaptation, Physiological↗
PubMed2006

MPB2C, a microtubule-associated plant factor, is required for microtubular accumulation of tobacco mosaic virus movement protein in plants.

Movement protein binding 2C (MPB2C) is a plant endogenous microtubule-associated protein previously identified as an interaction partner of tobacco (Nicotiana tabacum) mosaic virus movement protein (TMV-MP). In this work, the role of MPB2C in cell-to-cell transport of TMV-MP, viral spread of TMV, and subcellular localization of TMV-MP was examined. To this end, plants with reduced MPB2C levels were generated by a gene-silencing strategy. Local and systemic spread of TMV and cell-to-cell movement of TMV-MP were unimpaired in MPB2C-silenced plants as compared to nonsilenced plants, indicating that MPB2C is not required for intercellular transport of TMV-MP itself or spread of TMV. However, a clear change in subcellular distribution of TMV-MP characterized by a nearly complete loss of microtubular localization was observed in MPB2C-silenced plants. This result shows that the MPB2C is a central player in determining the complex subcellular localization of TMV-MP, in particular its microtubular accumulation, a phenomenon that has been frequently observed and whose role is still under discussion. Clearly, MPB2C mediated accumulation of TMV-MP at microtubules is not required for intercellular spread but may be a means to withdraw the TMV-MP from the cell-to-cell transport pathway.

Amino Acid Sequence↗
PubMed1968

Efficiency of nitrous acid as an inactivating and mutagenic agent of intact tobacco mosaic virus and its isolated nucleic acid.

When tobacco mosaic virus (TMV) and its isolated nucleic acid (TMV-RNA) were treated with nitrous acid, the nucleic acid was inactivated six times faster than the intact virus. Inactivation of both the infectious entities was exponential with treatment time to 0.1% level of survival. Eight different mutant phenotypes were scored after inactivation of TMV and TMV-RNA to 50, 10, 1.0, and 0.1% survival levels. Significantly more mutants in relation to unaltered isolates were induced at all levels of survival upon nitrous acid treatment of TMV than of TMV-RNA. Furthermore, the proportion of two specific mutant phenotypes was significantly greater in treated TMV than in treated TMV-RNA. No qualitative differences, however, were observed between the mutational spectra of nitrous acid-treated TMV and TMV-RNA. These results indicate that, in the intact virus, the viral capsid protects some of the sites involved in lethality; thus, proportionately more mutants are induced on nitrous acid treatment of TMV versus TMV-RNA.

Chemical Phenomena↗
PubMed1998

Chaperone protein GrpE and the GroEL/GroES complex promote the correct folding of tobacco mosaic virus coat protein for ribonucleocapsid assembly in vivo.

Several prokaryotic chaperone proteins were shown to promote the correct folding and in vivo assembly of tobacco mosaic virus coat protein (TMV CP) using a chimaeric RNA packaging system in control or chaperone-deficient mutant strains of Escherichia coli. Mutations in groEL or dnaK reduced the amount of both total and soluble TMV CP, and the yield of assembled TMV-like particles, several-fold. Thus both GroEL and DnaK have significant direct or indirect effects on the overall expression, stability, folding and assembly of TMV CP in vivo. In contrast, while cells carrying a mutation in grpE expressed TMV CP to a higher overall level than control E. coli, the amounts of both soluble CP and assembled TMV-like particles were below control levels, suggesting a negative effect of GrpE on overall CP accumulation, but positive role(s) in CP folding and assembly. Curiously, cells with mutations in groES and, to a lesser extent, dnaJ expressed total, soluble and assembled forms of TMV CP significantly above control values, suggesting some form of negative control by these chaperone proteins. To avoid pleiotropic effects or artefacts in chaperone-null mutants, selected chaperone proteins were also over-expressed in control E. coli cells. Overproduction of GroEL or GroES alone had little effect. However, co-overexpression of GroEL and GroES resulted in a two-fold increase in soluble TMV CP and a four-fold rise in assembled TMV-like (pseudovirus) particles in vivo. Moreover, TMV CP was shown to interact directly with GroEL in vivo. Together, these results suggest that GrpE and the GroEL/GroES chaperone complex promote the correct folding and assembly of TMV CP into ribonucleocapsids in vivo.

Bacterial Proteins↗
PubMed1987

Coat protein gene sequence of tobacco mosaic virus encodes a host response determinant.

The common strain and tomato strain of tobacco mosaic virus (TMV) are known to be closely related to each other. However, plants with the N' gene, such as Nicotiana sylvestris and Nicotiana tabacum L. cv. Bright Yellow, respond differently to infections by these viruses. In the N' plants, TMV-OM (common strain) spreads systemically with mosaic symptoms, whereas TMV-L (tomato strain) induces the necrotic response of plants, causing local lesions. To reveal the viral factor of TMV-L inducing the necrotic response, we have constructed several recombinant viruses between the two strains, in which TMV-L RNA was partly replaced by TMV-OM RNA. The recombinant viruses having the coat protein gene sequence of TMV-OM in place of TMV-L produced no necrotic local lesions but spread systemically with mosaic symptoms in the N' plants. On the other hand, the recombinant viruses having TMV-OM-derived sequences other than the coat protein gene sequence, and in which the coat protein gene sequence of TMV-L still remained, produced necrotic local lesions. These observations indicate that the viral factor of TMV-L responsible for the necrotic response of the N' plants is coded in the coat protein gene sequence.

Base Sequence↗
PubMed1999

Protective immunity against murine hepatitis virus (MHV) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an MHV epitope.

Hybrids of tobacco mosaic virus (TMV) were constructed with the use of fusion to the coat protein peptides of 10 or 15 amino acids, containing the 5B19 epitope from the spike protein of murine hepatitis virus (MHV) and giving rise to TMV-5B19 and TMV-5B19L, respectively. The TMV hybrids were propagated in tobacco plants, and the virus particles were purified. Immunogold labeling, with the use of the monoclonal MAb5B19 antibody, showed specific decoration of hybrid TMV particles, confirming the expression and display of the MHV epitope on the surface of the TMV. Mice were immunized with purified hybrid viruses after several regimens of immunization. Mice that received TMV-5B19L intranasally developed serum IgG and IgA specific for the 5B19 epitope and for the TMV coat protein. Hybrid TMV-5B19, administered by subcutaneous injections, elicited high titers of serum IgG that was specific for the 5B19 epitope and for coat protein, but IgA that was specific against 5B19 was not observed. Mice that were immunized with hybrid virus by subcutaneous or intranasal routes of administration survived challenge with a lethal dose (10 x LD50) of MHV strain JHM, whereas mice administered wild-type TMV died 10 d post challenge. Furthermore, there was a positive correlation between the dose of administered immunogen and protection against MHV infection. These studies show that TMV can be an effective vaccine delivery vehicle for parenteral and mucosal immunization and for protection from challenge with viral infection.

Administration, Intranasal↗
PubMed1991

Biological activities of hybrid RNAs generated by 3'-end exchanges between tobacco mosaic and brome mosaic viruses.

Sequences within the conserved, aminoacylatable 3' noncoding regions of brome mosaic virus (BMV) genomic RNAs 1, 2, and 3 direct initiation of negative-strand synthesis by BMV polymerase extracts and, like sequences at the structurally divergent but aminoacylatable 3' end of tobacco mosaic virus (TMV) RNA, are required in cis for RNA replication in vivo. A series of chimeric RNAs in which selected 3' segments were exchanged between the tyrosine-accepting BMV and histidine-accepting TMV RNAs were constructed and their amplification was examined in protoplasts inoculated with or without other BMV and TMV RNAs. TMV derivatives whose 3' noncoding region was replaced by sequences from BMV RNA3 were independently replication competent when the genes for the TMV 130,000-M(r) and 180,000-M(r) replication factors remained intact. TMV replicase can thus utilize the BMV-derived 3' end, though at lower efficiency than the wild-type (wt) TMV 3' end. Providing functional BMV RNA replicase by coinoculation with BMV genomic RNAs 1 and 2 did not improve the amplification of these hybrid genomic RNAs. By contrast, BMV RNA3 derivatives carrying the 3' noncoding region of TMV were not amplified when coinoculated with wt BMV RNA1 and RNA2, wt TMV RNA, or all three. Thus, BMV replicase appeared to be unable to utilize the TMV 3' end, and there was no evidence of intervirus complementation in the replication of any of the hybrid RNAs. In protoplasts coinoculated with BMV RNA1 and RNA2, the nonamplifiable RNA3 derivatives bearing TMV 3' sequences gave rise to diverse new rearranged or recombined RNA species that were amplifiable.

Cloning, Molecular↗
PubMed2005

Comparative effects of salmeterol, albuterol, and ipratropium on normal and impaired mucociliary function in sheep.

STUDY OBJECTIVE: We measured tracheal mucus velocity (TMV), a marker of mucociliary clearance (MCC), in sheep before and for 12 h after treatment with salmeterol, albuterol, ipratropium, or vehicle to determine the effects on normal MCC. We also determined if these agents could reverse the depression in TMV caused by inhaled human neutrophil elastase (HNE), a model of abnormal MCC. METHODS: Study 1: TMV was measured initially and then for 6 h after metered-dose inhaler treatment with salmeterol (42 microg), albuterol (180 microg), ipratropium bromide (36 microg), or vehicle. After 6 h, the sheep in the albuterol and ipratropium treatment arms were administered a second dose of drug, whereas the salmeterol and vehicle treatment arms received vehicle. TMV was measured for another 6 h. Study 2: Six sheep inhaled HNE aerosol, which significantly reduced TMV by 2 h. At this point, the sheep were treated with either salmeterol, albuterol, ipratropium, or vehicle, and the effects on TMV were measured for another 6 h. This experiment was repeated in four sheep using only salmeterol and albuterol, but the posttreatment measurements were extended to 12 h. RESULTS: Study 1: Only salmeterol and albuterol increased TMV (p < 0.05) during the initial 6-h period. From 6 to 12 h only, the salmeterol-treated sheep had TMV that remained at or above the initial TMV for the entire time, although both albuterol and ipratropium showed enhancement of TMV compared to vehicle. Study 2: Salmeterol and albuterol reversed the HNE-induced depression in TMV to a similar degree over the 6-h time course. However, the protection afforded by salmeterol was more prolonged than that seen with albuterol if the posttreatment interval was extended to 12 h. Ipratropium and vehicle had no effect. CONCLUSION: We conclude that salmeterol and albuterol can stimulate normal MCC and reverse HNE-induced mucociliary dysfunction and that salmeterol has a longer duration of action in these models of normal and abnormal MCC.

Adrenergic beta-Agonists↗
PubMed1996

Tobamovirus cross protection using a potexvirus vector.

Cross protection is the ability of one virus to prevent or delay infection by a related challenge virus. To examine this phenomena, a potato X potexvirus (PVX) vector (Chapman et al., 1992, Plant J. 2, 549) was used to systemically express the tobacco mosaic tobamovirus (TMV) coat protein (CP) open reading frame in Nicotiana benthamiana. PVX constructs induce mild mosaic symptoms in N. benthamiana, whereas TMV infection results in rapid systemic necrosis and plant death. Healthy plants or plants preinfected with the unmodified PVX vector succumbed to necrosis within 1 week of TMV challenge inoculation. However, plants preinfected with PVX vectors expressing the TMV CP or RNA coding sequence displayed a 1- to 2-week delay in the appearance of TMV-induced necrosis along with reduced accumulations of TMV. Symptom delay and reductions in the accumulation of TMV were more pronounced in plants protected with the vector expressing the TMV CP than with the vector expressing a nontranslatable CP sequence. Protection was overcome more quickly using a 10-fold higher concentration of challenge inoculum and was ineffective against TMV RNA or a TMV construct expressing a distantly related tobamovirus CP. These findings demonstrate that both RNA and protein are involved in this cross protection phenomenon, but that CP is the main contributor to protection. Taken together, these studies indicate that virus vectors can be used to simulate both cross protection and transgene-derived coat protein-mediated protection, thus providing a new method to further investigate the mechanisms behind each.

Capsid↗
PubMed1999

Limitations to tobacco mosaic virus infection of turnip.

Turnip vein-clearing virus (TVCV) and tobacco mosaic virus (TMV) represent subgroups of tobamoviruses infecting cruciferous and solanaceous plants, respectively. To identify adaptations that may have been necessary in the evolution of the TVCV subgroup from a TMV-like ancestor, the infection of turnip plants by TMV and by chimeras between TMV and TVCV was explored. TMV accumulated at spatially limited sites on inoculated turnip leaves as determined by leaf skeleton hybridization. A plasmid DNA containing a complete TVCV cDNA, when transcribed in vitro, produced RNA that was infectious to tobacco and turnip plants. TVCV-TMV chimeric genomes with junctions within coding regions were not infectious to tobacco, though the movement protein (MP) chimera was infectious to tobacco with a TMV MP transgene. Reciprocal chimeras with junctions between genes were infectious to tobacco. TVCV with a TMV MP gene infected turnips. The other tested chimeras were not detected in non-inoculated leaves, but were found in the inoculated leaves. Thus, the TMV MP is not responsible for the limitation of TMV spread in turnips.

Brassica↗
PubMed1953

Relationships between tobacco mosaic virus biosynthesis and the nitrogen metabolism of the host.

1. Comparisons of the nitrogen content of TMV-infected and uninfected tobacco leaf discs at various times after inoculation show that virus synthesis is associated with a net increase in protein content. This excess protein is due to: (a) TMV, (b) an excess in insoluble protein which develops soon after inoculation and ends about 100 hours before cessation of TMV synthesis, and (c) an excess in soluble non-virus protein, which is variable in size and which only occurs during the time of virus synthesis. A deficiency in non-protein nitrogen occurs during the time when virus appears. 2. Isotope experiments with N(15)-labelled nutrient show that: (a) The bulk of TMV nitrogen is derived from the free ammonia of the host tissue. (b) Amino acid residues of TMV protein are not derived from the corresponding free amino acids in the host. (c) The appearance of TMV is preceded by the synthesis of an insoluble precursor of the virus which is then converted into TMV or some soluble intermediate protein. This effect is associated with a cell particulate which represents a small fraction of the total insoluble protein. (d) Infected tissue synthesizes de novo small amounts of soluble non-virus protein, which may represent intermediates in TMV synthesis. (e) Infected tissue fails to synthesize a rapidly turned-over soluble protein which is synthesized in comparable uninfected tissue. (f) TMV synthesis is preceded by a temporary enhancement of the metabolic stability of an insoluble protein component. 3. The results lead to the conclusion that TMV formation is due to diversion of some part of the host's protein-synthesizing apparatus from its normal course.

Nitrogen↗
PubMedDate not supplied

Broad resistance to tobamoviruses is mediated by a modified tobacco mosaic virus replicase transgene.

Tobacco plants made transgenic to express the wild type tobacco mosaic virus (TMV) 183-kDa replicase gene were not resistant to TMV. However, transgenic plants containing essentially the same sequences, but with an additional insertion that would terminate translation in the middle of the 183-kDa gene, were highly resistant to systemic infection by TMV and other tobamoviruses. The 1.4-kbp insertion in the replicase open reading frame (ORF) of the resistant plants was shown by DNA sequencing to be an IS10-like transposable element, which apparently inserted itself into the TMV sequence at nucleotide 2875 sometime during the propagation of this replicase ORF plasmid (pREP21). Because of four stop codons, in frame with the TMV replicase ORF on the immediate 5' border of the IS insertion, REP21 effectively represents domain 1 (putative methylase domain) and a portion of domain 2 (putative helicase domain) of the TMV replicase ORF. REP21 Xanthi tobacco plants had a level of resistance to TMV similar to other reported transgenic replicase plants. No TMV was detected in upper leaves of these plants at 1-mo postinoculation. In addition, REP21 plants were resistant to an unusually broad range of tobamoviruses including tomato mosaic virus, tobacco mild green mosaic virus, TMV-U5, green tomato atypical mosaic virus, and ribgrass mosaic virus. These plants were not resistant to cucumber mosaic cucumovirus. The lack of systemic infection by TMV was due to reduced multiplication in inoculated leaves rather than complete prevention of replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1987

Tobacco mosaic virus replicase and replicative structures.

The RNA-dependent RNA polymerase (replicase) mediating the replication of tobacco mosaic virus (TMV) has been investigated in a number of laboratories over a period of 20 years. Cell-free enzyme preparations have been prepared which can continue the synthesis of nascent complementary RNA, initiated in vivo; however, the enzyme does not require, nor does it respond to, exogenous viral RNA as a template. The presence in plants of a virus-stimulated, host-encoded RNA-dependent RNA polymerase (RdRp) has added confusion to this field; it is now generally conceded, however, that this enzyme is not the TMV replicase. Our recent studies have emphasized several aspects of TMV RNA replication. We have examined the nature of TMV replicative structures synthesized in vitro by utilizing a partially purified enzyme preparation isolated from TMV-infected tobacco tissue. Radiolabelled products of the reaction were analysed on agarose gels and fractions with the predicted electrophoretic migration and nuclease sensitivities of replicative form (RF) and replicative intermediate (RI) were isolated. These fractions were hybridized to a collection of bacteriophage M13 clones containing portions of the TMV genome of both plus and minus polarity. The nascent synthesis in the RI-like molecules was restricted to the plus viral strand, while the new synthesis in the RF-like molecules was of both plus and minus polarity. Solubilization of the membrane-bound replicase with the non-ionic detergent CHAPS has yielded complexes which remain in solution after high-speed centrifugation. The solubilized replication complexes have been utilized as starting material for enzyme purification by Sepharose 4B gel filtration chromatography. The intracellular site of synthesis of TMV RNA has been reinvestigated in the light of reports suggesting a nuclear site of replication. The conclusion for nuclear synthesis has been based on fractionation of subcellular homogenates of virus-infected leaves or mesophyll protoplasts and identification of virus-related proteins associated with these fractions. In our studies, however, we conclude that these procedures can be misleading in that the 126,000 Mr TMV protein (and replicase activity) were found in all fractions of the homogenate analysed. Double-stranded TMV RNA, on the other hand, was barely detectable in preparations of purified nuclei; instead it was concentrated in the post-nuclear supernatant, suggesting that the nucleus is not the site of TMV RNA synthesis.

Chromatography, Agarose↗
PubMedDate not supplied

[Synthesis of the tobacco mosaic virus in blocked spreading of infection].

The possibility of infection of tobacco upper leaves with tobacco mosaic virus (TMV) was examined in experiments where the inoculum was imbibed through the cut stem. The inoculum used were: a) a preparation of a virus-specific informosome-like ribonucleoproteins (vRNP) isolated from TMV-infected plants; b) a TMV preparation; or c) a mixture of TMV and vRNP. Multiplication of TMV in upper leaves was observed in neither of the variants; nevertheless in the vascular tissue and/or probably in adjoining parenchymal cells, two kinds of RNA were synthesized: of mol. w. (1.1--1.3) X 10(6) and (0.6--0.8) X 10(6). These RNA were not found in healthy plants in the presence of actinomycin D. The synthesis of genomic TMV RNA is suppressed under these conditions. Thus, some kind of abortive TMV infection takes place under the condition of experimental inoculation of plants through a cut stem. Molecular hybridization with the DNA of recombinant plasmid containing a nucleotide sequence complementary to the 3'-portion of genomic TMV RNA proves that short RNAs synthesized under the abortive infection conditions are TMV-specific. The experiments with differential temperature treatment of N-gene-containing plants under abortive infection conditions suggest that necrotization is not necessarily induced by genomic TMV RNA synthesis.

Molecular Weight↗