The proteins of mutants of TMV: composition and structure of chemically evoked mutants of TMV RNA.
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Amino Acids↗
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A TMV RNA nucleotide sequence specifically recognized by TMV protein.
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Base Sequence↗
Effect of diethyl pyrocarbonate on the biological activity of intact TMV and TMV-RNA.
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Adenine Nucleotides↗
Assembly of tobacco mosaic virus and TMV-like pseudovirus particles in Escherichia coli.
High-level expression of plant viral proteins, including coat protein (CP), is possible in Escherichia coli. Native tobacco mosaic virus (TMV) CP expressed in E. coli remains soluble but has a non-acetylated N-terminal Ser residue and following extraction, is unable to package TMV RNA in vitro under standard assembly conditions. Changing the Ser to Ala or Pro by PCR-mutagenesis did not confer assembly competence in vitro, despite these being non-acetylated N-termini present in two natural strains of TMV. All TMV CPs made in E. coli formed stacked cylindrical aggregates in vitro at pH 5.0 and failed to be immunogold-labelled using a mouse monoclonal antibody specific for helically assembled TMV CP. TMV self-assembly has been studied extensively in vitro, and an origin of assembly sequence (OAS) mapped internally on the 6.4 kb ssRNA genome. Pseudovirus particles can be assembled mono- or bi-directionally in vitro using virus-derived CP and chimeric ssRNAs containing the cognate TMV OAS, but otherwise of unlimited length and sequence. Studies on plant virus assembly in vivo would be facilitated by a model system amenable to site-directed mutagenesis and rapid recovery of progeny particles. When chimeric transcripts containing the TMV OAS were co-expressed with TMV CP in vivo for 2-18 h, helical TMV-like ribonucleoprotein particles of the predicted length were formed in high yield (up to 7.4 micrograms/mg total bacterial protein). In addition to providing a rapid, inexpensive and convenient system to produce, protect and recover chimeric gene transcripts of any length or sequence, this E. coli system also offers a rapid approach for studying the molecular requirements for plant virus "self-assembly" in vivo. Transcription of a full-length cDNA clone of TMV RNA also resulted in high levels of CP expression and assembly of sufficient intact genomic RNA to initiate virus infection of susceptible tobacco plants.
Capsid↗
The expression of the TMV-specific 30-kDa protein in tobacco protoplasts is strongly and selectively enhanced by actinomycin.
The TMV-encoded 30-kDa protein has been implicated in the cell-to-cell transport of TMV in the infected plant. The polyethylene glycol-mediated inoculation of tobacco protoplasts with TMV particles and TMV RNA was used to compare the time courses of the viral 30-kDa protein synthesis in vivo. Upon infection of protoplasts with TMV RNA, the synthesis of the viral 30-kDa protein starts after 4 to 6 hr, has its maximum after 8 to 10 hr, and decreases. After inoculation of protoplasts with TMV, however, the start of the viral 30-kDa protein synthesis and its maximum are delayed by 2 hr, followed by the same decrease. We show that actinomycin D dramatically stimulates the synthesis of the 30-kDa protein by up to 2 orders of magnitude, whereas the synthesis of the viral 126 kDa, the 183 kDa, and the coat protein is increased only by a factor of 2. Surprisingly, actinomycin V is twice as active as actinomycin D, whereas actinomycin I is nearly inactive. The specific stimulation of the 30-kDa synthesis by actinomycin D in vivo depends neither on the Nicotiana variety nor on the TMV strain used. Final evidence that the 30-kDa protein is truly TMV-derived is provided by the slightly different electrophoretic mobilities of the 30-kDa proteins encoded by TMV strains vulgare, dahlemense, and U2. The identification of the 30-kDa protein in two-dimensional gels was achieved for the first time by a combination of ionic and nonionic detergents for the solubilization of the 30-kDa protein and by the specific stimulation of its synthesis by actinomycin D. The mechanism of the strong and selective actinomycin effect on the viral 30-kDa protein synthesis in vivo is as yet obscure. Actinomycin does not appear to act directly on viral protein biosynthesis, since it neither stimulates the 30-kDa synthesis upon translation of TMV RNA in vitro nor alters the ratio of the products. Actinomycin may rather act by inhibiting selectively the synthesis of a host factor whose synthesis starts at least 4 hr after TMV infection and which strongly inhibits the expression of the viral 30-kDa transport protein.
Capsid Proteins↗
Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA.
We have constructed three tobacco mosaic virus (TMV) cDNA derivatives by modification of the full-length cDNA clone from which infectious TMV-RNA can be transcribed in vitro. A coatless TMV construct lacks most of the coat protein gene and chimeric TMV constructs retain the bacterial chloramphenicol acetyltransferase (CAT) gene in place of the coat protein gene. When in vitro transcripts from these cDNA derivatives were inoculated on the local lesion tobacco plants, TMV-specific lesions were produced. In the case of the TMV-CAT chimeras, however, the lesions were small compared to those of wild-type TMV and those produced by transcript derived from the coatless construct. Northern blot analysis of RNA extracted from the inoculated leaves of the systemic host plants revealed replication of the derivative genomic RNAs and production of their own subgenomic RNAs corresponding to the coat protein mRNA. The TMV-CAT chimeras produced biologically active CAT in the inoculated leaves of the systemic host. CAT activity increased at least until 2 weeks post-inoculation and was approximately 0.1 units/mg of tissue at 10 days post-inoculation. Thus, TMV-RNA may be utilized as a new plant expression vector.
Blotting, Northern↗
Resistance to TMV in transgenic plants results from interference with an early event in infection.
Constitutive expression of the tobacco mosaic virus (TMV) coat protein (CP) gene in transgenic tobacco plants results in inhibition of disease symptom development following inoculation with TMV. Evidence is presented here that this protection is also observed in leaf mesophyll protoplasts isolated from these plants. Protoplasts were resistant to infection by TMV at concentrations of 10 microgram/ml to 1 mg/ml when introduced by either electroporation or polyethylene glycol-mediated inoculation. There was little protection against infection by TMV RNA and the protection was lost as the concentration of TMV RNA in the inoculum increased. When virus was incubated briefly at pH 8.0 prior to inoculation, protection broke down in a manner similar to that observed following RNA inoculation. Analogous results were obtained in experiments with whole plants. Because virus treated in this manner has presumably lost little or no CP, these results suggest that expression of the TMV CP gene in transgenic plant cells prevents TMV from uncoating. A model is presented for the mechanism of this blockage which relates these results to early events in TMV infection.
Capsid↗
Assessment of the effectiveness of a nuclear-launched TMV-based replicon as a tool for foreign gene expression in plants in comparison to direct gene expression from a nuclear promoter.
An environmentally safe Tobacco Mosaic Virus (TMV)-based expression replicon was constructed that lacks movement protein (MP) and coat protein (CP), and which expresses the green fluorescent protein (GFP) gene from a full CP subgenomic promoter. The TMV replicon, whose cDNA was positioned between an enhanced Cauliflower Mosaic Virus 35S promoter (CaMV) and a self-cleaving hammerhead ribozyme with a downstream nopaline synthase gene polyadenylation signal [nos-poly(A)], was assessed for its effectiveness to accumulate GFP upon agroinfiltration into plant leaves compared to a control construct in which GFP was directly expressed from the enhanced CaMV 35S promoter. It was determined that individually expressing cells produced ca. 9-fold more GFP from the TMV-based replicon than from the enhanced 35S promoter. In contrast, GFP measurements from total leaf extracts determined that leaves infiltrated with the TMV-based replicon produced ca. 7-fold less GFP than the control construct. These apparently contradictory results can be explained by the low infectivity of the TMV-based replicon as it was found that the number of foci expressing GFP produced in leaves agroinfiltrated with the TMV-based replicon was ca. 66-fold lower than produced by the control.
Cell Nucleus↗
Selective encapsidation of CAT gene transcripts in TMV-infected transgenic tobacco inhibits CAT synthesis.
Young tobacco seedlings (F1-progeny), transformed to express chloramphenicol acetyltransferase (CAT) mRNA with or without a 3'-proximal copy of the origin-of-assembly sequence (OAS) from tobacco mosaic virus (TMV) RNA (residues 5118-5550), were inoculated with TMV. After 21 days, virus symptoms were observed and systemic TMV infections were confirmed by Western blotting for viral coat protein and by electron microscopy of leaf saps. CAT activities were measured in extracts of leaf discs taken before, and 21 days after, virus inoculation. On average, the systemic leaves from TMV-infected CAT-transgenic plants containing the OAS exhibited 3.2-fold less CAT activity than the equivalent leaves from CAT-transgenic control plants lacking the OAS. Hence selective, OAS-dependent encapsidation of nuclear DNA transcripts into TMV-like (pseudovirus) particles can reduce expression of a particular mRNA, post-transcriptionally, in vivo. Furthermore, these data indicate that TMV self-assembly is not restricted to an exclusive subcellular compartment in vivo, and that formation of natural pseudovirions (A. Siegel, Virology 46, 50-59 (1971)) may shut off specific host RNA functions.
Acetyltransferases↗
TMV-peptide fusion vaccines induce cell-mediated immune responses and tumor protection in two murine models.
Fusion of peptides to viral carriers has proven an effective method for improving cellular immunity. In this study we explore the ability of a plant virus, Tobacco mosaic virus (TMV), to stimulate cellular immunity by interacting directly with immune cells. Fluorescently labeled TMV was incubated in vitro with murine spleen or lymph node cells, and near quantitative labeling of lymphocytes was achieved after 2 h, which persisted for up to 48 h. Direct TMV uptake and upregulation of the CD86 activation marker was measured in nearly all dendritic cells (DCs) by flow cytometry. To demonstrate that TMV can also provide functional antigen delivery and immune stimulation in vivo, two well-characterized T-cell epitopes that provide protection against tumor challenge in mice were fused to TMV coat protein by genetic manipulation, or by chemical conjugation. Vaccination of C57BL/6 mice elicited measurable cellular responses by interferon gamma (IFN gamma) ELISpot and resulted in significantly improved protection from tumor challenge in both the EG.7-Ova and B16 melanoma models. From these results we conclude that TMV was an effective antigen carrier for inducing cellular immune responses to less than 1 microg of peptide.
Animals↗
The helicase domain of the TMV replicase proteins induces the N-mediated defence response in tobacco.
Tobacco mosaic virus (TMV) induces the hypersensitive response (HR) in tobacco plants containing the N gene. This defence response is characterized by cell death at the site of virus infection and inhibition of viral replication and movement. A previous study indicated that a portion of the TMV replicase containing a putative helicase domain is involved in HR induction. Here, this observation is confirmed and extended by showing that non-viral expression of a 50 kDa TMV helicase fragment (p50) is sufficient to induce the N-mediated HR in tobacco. Like the HR elicited by TMV infection, transgenic expression of p50 induces a temperature-sensitive defence response. We demonstrate that recombinant p50 protein has ATPase activity, as suggested by the presence of conserved sequence motifs found in ATPase/helicase enzymes. A point mutation that alters one of these motifs abolishes ATPase activity in vitro but does not affect HR induction. These results suggest that features of the TMV helicase domain, independent of its enzymatic activity, are recognized by N-containing tobacco to induce TMV resistance.
Adenosine Triphosphatases↗
Selective recovery of foreign gene transcripts as virus-like particles in TMV-infected transgenic tobaccos.
A short origin-of-assembly sequence (OAS) located in the 30kDa movement protein gene, about 1.0kb from the 3'-end of the common strain of tobacco mosaic virus (TMV) RNA, nucleates encapsidation of the 6395-nucleotide-long genome by TMV coat protein in vitro, and presumably also in vivo. Single-stranded RNAs containing a foreign reporter gene sequence and the TMV OAS at their 5' - and 3' -ends, respectively, can be synthesized in vitro from recombinant SP6-transcription plasmids and will assemble spontaneously in vitro to form TMV-like 'pseudovirus' particles. In this paper, we show that foreign gene transcripts derived from the nuclear DNA of plants transformed by Agrobacterium tumefaciens, and which contain the TMV OAS, can be assembled into stable 'pseudovirus' particles in vivo during a systemic infection by TMV (helper). This is the first report of structural complementation between a heritable function bestowed on a transgenic plant and an infecting virus. As a route to protect, accumulate and recover a specific mRNA in vivo, in transgenic plant cells, this novel approach may find wider applications in developmental plant molecular biology.
Cloning, Molecular↗
Specific encapsidation of fragments of TMV RNA.
The in vitro reconstitution of tobacco mosaic virus (TMV) is initiated by the binding of a disk of TMV protein to the 'disk recognition site', a region of the RNA chain at or near the 5'-terminus for which the disk has special affinity. In order to gain insight into the recognition process, we have studied the ability of disks to encapsidate short RNA fragments produced by partial pancreatic or T1 RNase digestion of TMV RNA. The disk is capable of dicriminating among such fragments, encapsidating only a few of the many present in the digest. The products of encapsidation are short nucleoprotein rods of the same diameter as TMV and of length proportional to that of the encapsidated RNA fragment. The particles differ from TMV, however, in one significant aspect (apart from their length): they possess rings of RNA-free protein at one or both extremities of the rod. In the case of T1 RNase digestion the principal encapsidated fragments were fragments T1 (105 nucleotides) and a family of smaller fragments containing elements of the same sequence. Partial digestion with pancreatic RNase generated only one major fragment (fragment P1; 150 nucleotides) with affinity for the disk. Fragment T1 has been sequenced and shown to represent a portion of the coat protein cistron. Fragment P1 has been partially sequenced but its function is not yet known. Several lines of evidence indicate that fragment T1 is not the disk recognition site. The portion of the TMV RNA chain from which fragment P1 is derived, on the other hand, is encapsidated early in the reconstitution process; thus fragment P1 may contain the disk recognition site. Fragment T1 and fragment P1 both have purine-rich and cytosine-poor sequences near their termini. In addition, fragment T1, and possibly fragment P1, possess a periodicity of order three in purine residues. It seems likely that one or both of the aforesaid properties are largely responsible for the affinity of these fragments for the disk.
RNA, Viral↗
The effect of multiple dispersed copies of the origin-of-assembly sequence from TMV RNA on the morphology of pseudovirus particles assembled in vitro.
Supercoiled plasmid DNA was used as a template to transcribe long concatameric RNA molecules containing several dispersed copies of the origin-of-assembly sequence (OAS) from tobacco mosaic virus (TMV) RNA. When incubated with TMV coat protein "disks" in vitro, these RNAs spontaneously assembled into TMV-like pseudovirus particles. However, as each OAS initiated assembly more or less simultaneously, the concatameric RNA species generated complex nucleoprotein structures of predictable morphology. Similar structures were proposed some time ago (M. E. Taliansky, I. B. Kaplan, L. V. Yarvekulg, T. I. Atabekova, A. A. Agranovsky, and J. G. Atabekov, 1982, Virology 118, 309-316) to account for the RNase-sensitive phenotype of a ts mutant of TMV, Ni2519. These results extend the utility of our RNA-packaging vector system and confirm many of the predictions based on the current model for the self-assembly of TMV.
Base Sequence↗
The interaction between tylophorine B and TMV RNA.
Tylophorine B exhibits 60% inhibition against tobacco mosaic virus (TMV) at a concentration of 1.0 x 10(-6) g/ml. In our study, high affinity for TMV RNA and assembly origin of TMV RNA (oriRNA) was revealed, accompanied by the conformational change of RNA. Considering that TMV assembly begins with the specific recognition by the coat protein aggregate of oriRNA, and that tylophorine B has favorable interaction with oriRNA, we speculate that tylophorine B likely exerts its virus inhibition by binding to oriRNA and interfering with virus assembly initiation. This work may shed light on the possible molecular inhibition mechanism against TMV by tylophorine B, and provide clues in rational design of sequence-specific RNA binding antivirus drugs.
Alkaloids↗
Morphology and stability changes of recombinant TMV particles caused by a cysteine residue in the foreign peptide fused to the coat protein.
In the studies of expressing various foreign peptides using a TMV-based vector, a portion of morphologically altered progeny viral particles from some recombinant TMV constructs were detected by transmission electron microscopy in the first systematically infected upper leaves, but not in the fully expanded inoculated leaves, from infected tobacco plants. Furthermore, in vitro stability of such recombinant TMV constructs were lower than those of the wild type and other recombinant TMV constructs able to form regular rod-shape virions, hence causing the lower yields of recombinant viral particles purified from the infected tobacco plants. Our studies revealed that the presence of a cysteine residue in the foreign peptides, regardless of its position and the peptide sequence, was directly related to changes in the morphology and stability of these TMV recombinants.
Capsid Proteins↗
TMV recombinants encoding fused foreign transmembrane domains to the CP subunit caused local necrotic response on susceptible tobacco.
With regard to the effects of various foreign peptides fused to the coat protein subunits on the infectivity of corresponding TMV recombinants, some of TMV recombinants were found to induce necrotic local lesions on the inoculated leaves of susceptible tobacco. This paper reported that there existed a group of TMV recombinants in which the fused foreign peptides contained a transmembrane domain according to the predictions by three programs of SOSUI, TMpred and DAS. Further studies showed for the first time that a foreign transmembrane domain in a fused peptide of the corresponding TMV recombinant would result in the local lesions on the susceptible tobacco leaves. In addition, it was concluded that none of the TMV recombinants that systematically infected susceptible tobacco contained a transmembrane domain in the coat protein subunits.
Amino Acid Sequence↗
H protein, a minor protein of TMV virions, contains sequences of the viral coat protein.
H protein, a minor protein found associated with virions of tobacco mosaic virus (TMV) at an average of about one copy per virion and previously believed to be host-coded (Asselin and Zaitlin, 1978, Virology 91, 173-181), has been shown to contain sequences of the viral capsid protein. Two-dimensional tryptic peptide maps of 125I-labeled H protein (Mr 26,500) and coat protein (Mr 17,500) from TMV strains U1 and Dahlemense show that the respective H proteins contain most if not all of the labeled peptides of the coat proteins in addition to 2-3 unique peptides. The H proteins also contain unique antigenic determinants, as antibodies can be isolated which react strongly with the H protein but not with the coat protein of Dahlemense TMV. Finally, amino acid composition analysis of the U1-TMV H protein has shown the presence of methionine and histidine, amino acids not present in the coat protein of that strain. H protein appears to contain the same NH2 terminus as coat protein, as there is an H protein tryptic peptide that both comigrates in a two-dimensional system and produces the same acid cleavage product as the NH2-terminal tryptic peptide of coat protein. H protein also seems to have the same COOH terminus as coat protein, as cyanogen bromide digestion of Dahlemense-TMV coat protein and H protein indicates that each has a methionine about 12 amino acids from one terminus (known to be the COOH terminus of the coat protein). Thus, H protein is not structurally equivalent to coat protein with an addition on either its NH2 or COOH terminus. However, H protein does not appear to be a noncovalent aggregate of coat protein and some other protein. Rather, the model we favor for H protein structure is that of a branched fusion product between coat protein and another polypeptide of host or viral origin.
Capsid Proteins↗