Tobacco Mosaic Virus Mutant Research Articles

In vitro assembly of Tobacco mosaic virus coat protein variants derived from fission yeast expression clones or plants

Tobacco mosaic virus (TMV) derivatives are explored currently extensively with regard to nanotechnological applications. Since certain technically desired TMV mutants may not be accessible from plants, the utility of the fission yeast Schizosaccharomyces pombe for heterologous production of TMV coat protein (CP) variants was explored, including wild-type (wt) CP and two genetically engineered mutants: TMV-CP-His 6 containing a C-terminal hexahistidine (His 6) tag, and TMV-CP-E50Q with enhanced lateral CP subunit interactions. After establishing expression clones and protocols for enrichment of the CP variants, their ability to reconstitute TMV-like nanostructures in the presence or absence of RNA was tested in comparison with the corresponding plant-derived CP variants, which were expressed from infectious TMV constructs. Both TMV-CP-E50Q and TMV-CP-wt yielded TMV-like rods, irrespective of the proteins’ source. In contrast, His-tagged CP from plants produced only short rods in an inefficient manner, and no rods at all when expressed in yeast. This study introduces a novel approach to produce assembly competent TMV CP, but also demonstrates its limitations.

A quantitative analysis of complementation of deleterious mutants in plant virus populations

Complementation can be defined as the process by which the function affected by a mutation is provided in trans by fully competent genotypes. Complementation can, thus, counter the effects of selection of deleterious mutants. Complementation of mutants defective for replication, movement and transmission has been often described in experiments with viruses and, occasionally, has been reported to occur in their natural populations. However, the role of complementation in virus evolution has been overlooked. Here is provided a quantitative estimate of the efficiency of complementation, defined as the probability that a non-functional mutant accomplishes a function relative to a functional one. For this, the frequency of mutants of Tobacco mosaic virus (TMV) defective for cell to cell movement was estimated in wild type tobacco plants and in transgenic plants expressing the TMV movement protein (MP) from a transgene. Mutants lethal for cell-to-cell movement were complemented by wild-type TMV in the first case, and by the transgene-expressed MP in the second case. Assuming that complementation is fully efficient in the transgenic plants, a value for the efficiency of complementation of 0.34 was obtained. Thus, complementation can efficiently counter selection on lethal mutants, and may have an important role on virus evolution. Complementation may be relevant for management of viral diseases if the complemented deleterious mutation is linked to other functions affecting the pathogenicity or epidemiology of the virus.

Evidence for contribution of an internal ribosome entry site to intercellular transport of a tobamovirus.

Previously, it has been shown that tobacco mosaic virus (TMV) U1 and crucifer-infecting TMV contain a 75 nt internal ribosome entry site (IRES) upstream of movement protein (MP) gene (IRES(U1)(MP,75) and IRES(CR)(MP,75), respectively). A movement-deficient TMV mutant, KK6, has been constructed previously [Lehto, K., Grantham, G. L. & Dawson, W. O. (1990). Virology 174, 145-157] by insertion of the second coat protein subgenomic promoter (CP SGP-2) upstream of the MP gene, in addition to the natural CP SGP-1. Here, the authors compare the efficiency of movement function expression by KK6 and a derivative, K86, obtained by insertion of IRES(CR)(MP,75) between the CP SGP-2 and MP genes resulting in restoration of IRES(CR)(MP,75) function in the 5'-untranslated sequence of the I(2) subgenomic RNA of K86. The data indicate that the efficiency of K86 movement was largely restored by this insertion, which was apparently due to the translation-enhancing ability of IRES(CR)(MP,75).

A Nuclear Localization Signal and a Membrane Association Domain Contribute to the Cellular Localization of the Tobacco Mosaic Virus 126-kDa Replicase Protein

A transient expression system using onion epidermal cells was used to investigate domains of the Tobacco mosaic virus (TMV) 126-kDa replicase protein involved in cellular localization. Initially, a nuclear localization signal (NLS), identified within the amino-terminus of the 126-kDa protein, was investigated for its functionality using fusion constructs containing the green fluorescent protein (GFP). Fusion of the amino-terminal 70 amino acids of the 126-kDa protein, containing the NLS, to a β-glucuronidase-GFP open reading frame (ORF), directed the accumulation of fluorescence to the nucleus. In contrast, similar constructs lacking the NLS or containing a mutated NLS sequence failed to accumulate within the nucleus. Additional investigations using GFP fusion constructs containing the first 178 or 388 amino acids of the 126-kDa protein also displayed nuclear localization. However, fusion constructs encoding the first 781 amino acids or the entire 126-kDa ORF did not accumulate within the nucleus but instead associated with the endoplasmic reticulum (ER), forming spot-like inclusions. Thus, a dominant ER association domain exists between amino acids 388 and 781 of the 126-kDa protein. Interestingly, a full-length 126-kDa GFP fusion construct encoding a nonfunctional NLS mutation also localized to the ER but did not form inclusions. Furthermore, a TMV mutant containing the same nonfunctional NLS mutation failed to replicate in protoplasts. Together these findings suggest that both the NLS and the ER retention domain contribute to the functional localization of the 126-kDa protein.

Mutants of tobacco mosaic virus with temperature-sensitive coat proteins induce heat shock response in tobacco leaves.

We analyzed, with respect to heat shock proteins (HSPs), systemically reacting tobacco leaves inoculated with Tobacco mosaic virus (TMV), wild-type vulgare, and temperature-sensitive coat protein (CP) mutants Ni 118 (P20L) and flavum (D19A), kept at 23 or 30 degrees C. HSP18 and HSP70 mRNAs and proteins were induced with temperature-sensitive CP mutants after 1 to 2 days at 30 degrees C. After 4 to 6 days, HSP70 was also induced at 23 degrees C. The induction of HSPs paralleled the amount of insoluble TMV CP in leaf extracts, indicating that denatured TMV CP by itself induces a heat-shock response.

Expression of human hepatitis C virus core antigen in tobacco plants by tobacco mosaic virus-based vector system

Tobacco mosaic virus (TMV) has the potential to highly express foreign gene. A novel TMV-basedin trans expression system was constructed. A TMV mutant TSHc had its coat protein replaced with hepatitis C virus (HCV) core antigen gene. Anotherr TMV mutant TSBD was replicase-defective. Coinfection of the two mutants could cause systemic infection in tobacco plants byin trans complementation of their functions. TSHc could effectively replicate and assemble to viral particles, which were a little longer than that of wild-type TMV. HCV core antigen was expressed in whole tobacco plants. A similar expression level of HCV core antigen was detected on serial passages, which suggested that this viral expression system be stable.

Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA.

Little is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.

Identification of viral mutants by mass spectrometry.

A method to identify mutations of virus proteins by using protein mass mapping is described. Comparative mass mapping was applied to a structural protein of the human rhinovirus Cys1199 --> Tyr mutant and to genetically engineered mutants of tobacco mosaic virus. The information generated from this approach can rapidly identify the peptide or protein containing the mutation and, in cases when nucleic acid sequencing is required, significantly narrows the region of the genome that must be sequenced. High-resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and tandem mass spectrometry were used to identify amino acid substitutions. This method provides valuable information for those analyzing viral variants and, in some cases, offers a rapid and accurate alternative to nucleotide sequencing.

Transgenic Plants Expressing Potato Virus X ORF2 Protein (p24) Are Resistant to Tobacco Mosaic Virus and Ob Tobamoviruses

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.

Invasion of minor veins of tobacco leaves inoculated with tobacco mosaic virus mutants defective in phloem-dependent movement.

To fully understand vascular transport of plant viruses, the viral and host proteins, their structures and functions, and the specific vascular cells in which these factors function must be determined. We report here on the ability of various cDNA-derived coat protein (CP) mutants of tobacco mosaic virus (TMV) to invade vascular cells in minor veins of Nicotiana tabacum L. cv. Xanthi nn. The mutant viruses we studied, TMV CP-O, U1mCP15-17, and SNC015, respectively, encode a CP from a different tobamovirus (i.e., from odontoglossum ringspot virus) resulting in the formation of non-native capsids, a mutant CP that accumulates in aggregates but does not encapsidate the viral RNA, or no CP. TMV CP-O is impaired in phloem-dependent movement, whereas U1mCP15-17 and SNC015 do not accumulate by phloem-dependent movement. In developmentally-defined studies using immunocytochemical analyses we determined that all of these mutants invaded vascular parenchyma cells within minor veins in inoculated leaves. In addition, we determined that the CPs of TMV CP-O and U1mCP15-17 were present in companion (C) cells of minor veins in inoculated leaves, although more rarely than CP of wild-type virus. These results indicate that the movement of TMV into minor veins does not require the CP, and an encapsidation-competent CP is not required for, but may increase the efficiency of, movement into the conducting complex of the phloem (i.e., the C cell/sieve element complex). Also, a host factor(s) functions at or beyond the C cell/sieve element interface with other cells to allow efficient phloem-dependent accumulation of TMV CP-O.

Characterization of Deletion and Frameshift Mutants of Satellite Tobacco Mosaic Virus

A series of frameshift and deletion mutations was created in the genome of satellite tobacco mosaic virus (STMV) by modifying full-length cDNA clones of the type strain, from which biologically active transcripts could be synthesized in vitro. Deletions and frameshift mutations in the 5′ open reading frame had no effect, compared to wild-type STMV, on RNA accumulation, systemic movement, or the symptoms induced by STMV in Nicotiana tabacum co-inoculated with tobacco mild green mosaic tobamovirus (TMGMV). This implies that the protein encoded by this reading frame is not necessary for biological activity. Deletions and frameshift mutations in the coat protein open reading frame resulted in decreased accumulation of STMV RNA in N. tabacum, although these mutants were still capable of systemic movement, presumably in a nonencapsidated or free RNA form. Furthermore, the mild symptoms induced in tobacco by co-inoculations of wild-type STMV/TMGMV or infection with TMGMV alone were altered to severe systemic necrosis when plants were co-inoculated with these STMV coat protein mutants and TMGMV. Mutants within the 3′ untranslated region were much less able to accumulate in TMGMV-infected plants than was wild-type STMV, and under some growth conditions did not accumulate to detectable levels.

Studies of Coat Protein-Mediated Resistance to TMV: I. The PM2 Assembly Defective Mutant Confers Resistance to TMV

Tobacco mosaic virus mutant PM2 contains two amino acid changes in coat protein sequence relative to the sequence of the coat protein of TMV U1. This results in unstable infectivity, inability to cause normal systemic infection, and accumulation of elongated open helixes of coat protein. Using site-directed mutagenesis we demonstrated that the characteristics of PM2 are due to the change of Thr28 → Ile, while the second change, Glu95 → Asp, had no apparent effect on virion structure or infectivity. Transgenic Nicotiana tabacum cv Xanthi NN and Xanthi nn plants that accumulate coat protein that contains one or both of the amino acid changes are as resistant to TMV infection as transgenic plants that contain wildtype TMV coat protein. The implication of these results on a model for coat protein-mediated resistance that involves the interaction of transgenic coat protein with the challenge virus is discussed.

Reduction of tobacco mosaic virus accumulation in transgenic plants producing non-functional viral transport proteins.

Transgenic plants producing the 30K temperature-sensitive transport protein (TP) of tobacco mosaic virus (TMV) mutant Ni2519 (affecting cell-to-cell transport) were found to: (i) be susceptible to wild-type TMV U1 at 24 degrees C (a permissive temperature for Ni2519 TP), (ii) acquire a certain level of resistance to TMV U1 accumulation when maintained at 33 degrees C (a non-permissive temperature for Ni2519 TP) and (iii) lose the resistance to wild-type TMV after their transfer from 33 degrees C to 24 degrees C. It is suggested that reversible temperature-dependent conformational changes in Ni2519 TP are responsible for these phenomena and that production of a TP which is only partially functional in transgenic plants confers on these plants a resistance to the virus owing to reduction of the level of cell-to-cell transport. Transgenic tobacco plants producing the 32K TP of brome mosaic virus (BMV) acquired resistance to TMV U1 suggesting that BMV TP is partially functional in tobacco plants.

Wild-type coat protein gene of tobacco mosaic virus mutant Ni 2519.

Tobacco mosaic virus mutant Ni 2519 has a selected temperature-sensitive defect in the spreading of local lesions, and additional reported defects in virus assembly and in host range. Here we show that the temperature-sensitive local lesion-spreading defect (which maps in the assembly origin within the gene encoding protein p30) is probably independent of mutations in the pseudoassembly origin or of host range defects resulting from other mutations in the coat protein. One new host range mutant has been isolated.

Cis-acting elements for in trans complementation of replication-defective mutant of tobacco mosaic virus

We have shown that virus-encoded replicase components of tobacco mosaic virus (TMV) (130K/180K proteins) could complement a replication-defective mutant (LDR28) for viral replication in trans [Ogawa et al. Virology 185, 580–584 (1991)]. Using this trans-complementation system, we investigated the dispensability of regions of the 130K/180K protein genes as cis-acting elements for viral replication. A series of replication-defective mutants (LDRs) which had various deletions in the 130K/1 80K coding region were constructed. These were co-inoculated with a replication-competent mutant (LDCS29) into tobacco protoplasts. Accumulation of LDR-genomic RNA, CP mRNA, and CP was much increased by the removal of the 3′ terminal one-third of the readthrough part of 180K protein gene (residues 4529–4937 of TMV-RNA) from LDR, suggesting that this region interferes with the viral replication in this trans-complementation system. In addition, accumulation of CP mRNA and CP was much decreased by the removal of the 5′ terrminal half of the 30K protein gene (residues 4938–5263 of TMV-RNA), suggesting the presence of an element to enhance the synthesis of CP mRNA in this region.

Trans complementation of virus-encoded replicase components of tobacco mosaic virus

We examined whether the 130K and 180K proteins of tobacco mosaic virus (TMV), the putative virus-encoded replicase components, produced by a replication-competent TMV mutant could complement a replication-defective mutant in a single cell. The replication-competent mutant (LDCS29) had a deletion in the coat protein gene and the replication-defective mutant (LDR28) had a large deletion in the gene encoding the 130K and 180K proteins. Neither the replication of LDR28 nor the production of the coat protein from LDR28 or LDCS29 was detected when the mutants were inoculated separately into tobacco protoplasts. However, when the two mutants were co-inoculated, the production of the LDR28 genomic RNA and the subgenomic RNA for the coat protein and accumulation of the coat protein were observed. These results show that the virus-encoded replicase components of TMV complemented the replication-defective mutant in trans.

Plasmodesmatal function is probed using transgenic tobacco plants that express a virus movement protein.

A gene encoding a temperature-sensitive mutant (MPP154A) of the 30-kilodalton movement protein (MP) of tobacco mosaic virus (TMV) was transformed into Nicotiana tabacum cv Xanthi. Transgenic plants expressing the MPP154A gene complemented local and systemic movement of an MP-defective mutant of TMV (U3/12MPfs) at the permissive temperature of 24 degrees C but not at 32 degrees C, the nonpermissive temperature. A microinjection procedure was used to investigate the effects of the modified TMV MP on plasmodesmatal size-exclusion limits. Movement of fluorescein isothiocyanate-labeled dextran (F-dextran), with an average molecular mass of 9.4 kilodaltons, was detected between leaf mesophyll cells of the transgenic plants at 24 degrees C; however, no movement of either 3.9-kilodalton or 9.4-kilodalton F-dextrans was detected when the transgenic plants were held for 6 hours (or longer) at 32 degrees C. When these plants were shifted back to 24 degrees C for 6 hours, cell-to-cell movement of the F-dextrans was again observed. Accumulation of MPP154A was not affected by the temperature regime, nor was the subcellular distribution of the MP altered. These results are consistent with a change in the protein conformation of MPP154A at the nonpermissive temperature, which gives rise to a protein that fails to modify the molecular size-exclusion limits of plasmodesmata to the same extent as wild-type MP. Surprisingly, at 32 degrees C, movement of the F-dextrans was inhibited in transgenic plants expressing the wild-type MP gene; however, the inhibition was transient and was no longer detected after 48 hours at this elevated temperature. This transient inhibition of plasmodesmatal function was alleviated with Sirofluor, an inhibitor of callose ([1----3]-beta-D-glucan) synthesis. This result provides experimental evidence that callose deposition is involved in regulating the molecular size-exclusion limit of plasmodesmata in plants. Sirofluor had no effect on the inhibition of F-dextran movement at 32 degrees C in plants expressing the MPP154A gene, indicating that callose formation was not responsible for the failure of the temperature-sensitive mutant protein to alter the size-exclusion limit of plasmodesmata.

Complementation of coat protein-defective TMV mutants in transgenic tobacco plants expressing TMV coat protein

Transgenic tobacco plants ( Nicotiana tabacum cv. Xanthi) which express tobacco mosaic virus (TMV) U1 strain coat protein (CP) can complement both the assembly and the long-distance spread of CP-defective (DT1) or coat proteinless (DT1 G) mutants of TMV. Both mutants arose spontaneously from PM2 and exist only as unencapsidated RNA in the inoculated leaves of control tobacco plants, where they are unable to form virus particles or to spread systemically. TMV CP expressed in transgenic tobacco plants [CP + line 3404; P. Powell Abel, R. S. Nelson, B. De, N. Hoffmann, S. G. Rogers, R. T. Fraley, and R. N. Beachy, 1986, Science 232, 738–743 ] was able to package some of either mutant viral RNA into TMV-like particles in vivo and resulted in the long-range spread of infection. In vivo encapsidated DT1 RNA was recovered and reinoculated onto control or new CP + transgenic tobacco plants. Localized infection of control plants confirmed that no RNA recombination or reversion of the mutant RNA to wild-type had occurred during passage in the first CP + plant. In contrast, encapsidated DT1 RNA was unable to produce even local infection in CP + transgenic plants confirming that CP-mediated protection operates during the early stages of virus infection, including particle uncoating. By positive complementation, these results also confirm that TMV CP is required for the long-distance spread of infection.

Deletion of repeated sequences from tobacco mosaic virus mutants with two coat protein genes

Tobacco mosaic virus (TMV) hybrids were constructed in vitro with an extra coat protein gene (gene order 5′- 126K/183K gene-30K gene-first coat protein gene-subgenomic mRNA promoter:second coat protein gene-3′). One coat protein gene was wild type and the other was derived from one of three coat protein mutants that elicit distinctive phenotypic responses. The hybrids contained large sequence repeats (up to 747 nucleotides). RNA transcripts of each of seven hybrid constructs initiated infections in host plants, but the progeny virus contained only one coat protein gene with no additional or missing nucleotides. Progeny of mutants with the two functional coat protein genes in different order within the genome retained coat protein gene sequence predicted by the number of repeated nucleotides 3′ and 5′ of the differential nucleotides. In contrast, progeny that resulted from virus hybrids with a wild-type and a nonfunctional coat protein gene possessed only the wild-type coat protein gene, even when the predicted progeny was a free-RNA virus. This demonstrated that selection strongly affected which progeny became predominant in the resulting populations

Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus.

Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3 terminal nucleotides of the TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 micrograms/ml or 300 micrograms/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 micrograms/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to approximately 5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequence-specific RNA transcript of the expected size, but no protein product was detected.