Nucleotide sequence of Chinese rape mosaic virus (oilseed rape mosaic virus), a crucifer tobamovirus infectious on Arabidopsis thaliana

The complete nucleotide sequence of Chinese rape mosaic virus has been determined. The virus is a member of the tobamovirus genus of plant virus and is able to infect Arabidopsis thaliana (L.) Heynh systemically. The analysis of the sequence shows a gene array that seems to be characteristic of crucifer tobamoviruses and which is slightly different from the one most frequently found in tobamoviruses. Based on gene organization and on comparisons of sequence homologies between members of the tobamoviruses, a clustering of crucifer tobamoviruses is proposed that groups the presently known crucifer tobamovirus into two viruses with two strains each. A name change of Chinese rape mosaic virus to oilseed rape mosaic virus is proposed.Abbreviations 2-ME 2-mercaptoethanol - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - UTR untranslated region - MP movement protein - CP capsid protein - CRMV Chinese rape mosaic virus - TVCV turnip vein clearing virus - PaMMV paprika mild mottle virus - PMMV-I pepper mild mottle virus (Italian isolate) - PMMV-S pepper mild mottle virus (Spanish isolate) - ToMV tomato mosaic virus - TMV tobacco mosaic virus - TMGMV tobacco mild green mosaic virus - ORSV odontoglossum ringspot virus - SHMV sunn hemp mosaic virus - CGMMV cucumber green mottle mosaic virus - ORMV oilseed rape mosaic virus     

Polar uncoating of tobacco mosaic virus (TMV) with dimethylsulfoxide (DMSO) and subsequent reassembly of partially stripped TMV

Summary Increasing concentrations of dimethylsulfoxide (DMSO) strip tobacco mosaic virus (TMV) stepwise from the 3 end. The RNA tail increases in length up to 2,000 nucleotides (nu) reaching a region of very strong protein-RNA affinity. Thereafter, uncoating occurs from the other end and produces a second RNA tail 500 nu long. Further stripping of TMV proceeds from both ends, the long tail increasing in length up to 4,000 nu and the short one increasing more moderately and remaining below 2,000 nu. The region of strongest protein-RNA affinity is located between 4,000 and 5,000 nu away from the 3 end.Using the same conditions as for in vitro TMV reassembly, it is possible to recoat the RNA tails with viral protein preferentially in the 5 direction.The advantages of DMSO in studies of TMV protein-RNA interactions are discussed.     

Nucleotide sequence of a cloned cDNA copy of TMV (cowpea strain) RNA, including the assembly origin, the coat protein cistron, and the 3'' non-coding region

Summary The cloned cDNA derived from the 3 end of cowpea strain (Cc) RNA of tobacco mosaic virus (TMV) has been sequenced. Substantial sequence information of 1,060 nucleotides from the 3 end of the RNA reveals some interesting features: (1) the coat protein cistron corresponds to residues 210–701 from the 3 end. Some errors in the amino acid sequence previously reported have been corrected and the revised total length of the coat protein is 162 amino acid residues. The capping site of the coat protein mRNA is at residue 711 from the 3 end of genome RNA. (2) The assembly origin of reconstitution is positioned within the coat protein cistron at residue 369–461 which can be formed into a highly base-paired hairpin loop structure. The sequence, GAXGUUG, in the loop region and a triplet-repeated purine base tract surrounding the loop are found. These structural features are common to assembly origins of both Cc and vulgare strains. (3) We find the sequence highly homologous to, but distinct from, the genuine assembly origin. It will be called the pseudo-assembly origin, which is located in the corresponding region to the assembly origin of the vulgare strain, outside the coat protein cistron. There is also the sequence, GAXGUUG, in the middle of the region. (4) In the 5 flanking region of the coat protein cistron, a long reading frame, probably of 30 K protein, is found. The coding region is terminated in the coat protein cistron and thus the 30 K protein and the coat protein cistrons overlap. (5) The 3 non-coding region is 209 residues long and can be folded into a possible tRNA-like structure. Surprisingly, we find that the 3 terminal sequence of Cc RNA is not very similar to that of vulgare RNA but extensively homologous to that of turnip yellow mosaic virus (TYMV) RNA.     

The Complete Nucleotide Sequence of Odontoglossum Ringspot Virus (Cy-1 Strain) Genomic RNA

The complete nucleotide sequence of the genomic RNA of odontoglossum ringspot virus Cy-1 strain (ORSV Cy-1) was determined using cloned cDNA. This sequence is 6611 nucleotides long containing four open reading frames, which correspond to 126 K, 183 K, 31 K, and 18 K proteins. Its genomic organization is similar to other tobamoviruses, TMV-V(vulgare), TMV-L (tomato strain), tobacco mild green mosaic virus (TMGMV) and cucumber green mottle mosaic virus (CGMMV). The 5′ non-coding regions of ORSV Cy-1 is 62 nucleotides. The ORFs encoded a 126 K polypeptide and a 183 K read-through product in which helicase-sequence and polymerase-sequence motifs are found. The ORFs encoding the 126 K and 183 K proteins have 61% and 63% identities with those of TMV-V. The third ORF encoded a 31 K protein homologous to TMV cell-to-cell movement protein. It has 63% identities with that of TMV-V. The fourth ORF encoded an 18 K coat protein. The 5′ non-coding region, which extends from base 1 to 62 has 2 G residues and a ribosome binding site (AUU). The 3′ non-coding region, 414 nucleotides in length, is entirely different from that of other tobamoviruses.     

Molecular analysis of the capsid protein gene of a german isolate of barley mild mosaic virus

Summary Barley mild mosaic virus (BaMMV) is one of the agents causing the barley yellow mosaic disease. The sequence corresponding to the 3end of the BaMMV RNA1 of a German isolate was sequenced and the coding sequence for the 251 amino acid containing capsid protein was determined. Comparison of this sequence to other potyviral sequences and to the corresponding sequence of two Japanese isolates of BaMMV was done. The three different isolates of BaMMV show a high degree of similarity.Abbrevations BaMMV barley mild mosaic virus - BaYMV barley yellow mosaic virus; bp: base pair - IPTG isopropyl -D thiogalactopyranoside - kb kilo base - NTR nontranslated region - ORF open reading frame - PVDF polyvinylidene difluoride     

Subcellular localization and detection of <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> ORF6 protein by immunoelectron microscopy

Members of the genus Tobamovirus represent one of the best-characterized groups of plant positive, single stranded RNA viruses. Previous studies have shown that genomes of some tobamoviruses contain not only genes coding for coat protein, movement protein, and the cistron coding for different domains of RNA-polymerase, but also a gene, named ORF6, coding for a poorly conserved small protein. The amino acid sequences of ORF6 proteins encoded by different tobamoviruses are highly divergent. The potential role of ORF6 proteins in replication of tobamoviruses still needs to be elucidated. In this study, using biochemical and immunological methods, we have shown that ORF6 peptide is accumulated after infection in case of two isolates of Tobacco mosaic virus strain U1 (TMV-U1 common and TMV-U1 isolate A15). Unlike virus particles accumulating in the cytoplasm, the product of the ORF6 gene is found mainly in nuclei, which correlates with previously published data about transient expression of ORF6 isolated from TMV-U1. Moreover, we present new data showing the presence of ORF6 genes in genomes of several tobamoviruses. For example, in the genomes of other members of the tobamovirus subgroup 1, including Rehmannia mosaic virus, Paprika mild mottle virus, Tobacco mild green mosaic virus, Tomato mosaic virus, Tomato mottle mosaic virus, and Nigerian tobacco latent virus, sequence comparisons revealed the existence of a similar open reading frame like ORF6 of TMV.     

Tobacco mosaic virus particle structure and the initiation of disassembly

The structure of an intact tobacco mosaic virus (TMV) particle was determined at 2.9 A resolution using fibre diffraction methods. All residues of the coat protein and the three nucleotides of RNA that are bound to each protein subunit were visible in the electron density map. Examination of the structures of TMV, cucumber green mottle mosaic virus and ribgrass mosaic virus, and site-directed mutagenesis experiments in which carboxylate groups were changed to the corresponding amides, showed that initial stages of disassembly are driven by complex electrostatic interactions involving at least seven carboxylate side-chains and a phosphate group. The locations of these interactions can drift during evolution, allowing the viruses to evade plant defensive responses that depend on recognition of the viral coat protein surface.     

The sequence surrounding the translation initiation codon of the pea plastocyanin gene increases translational efficiency of a reporter gene

The 5-upstream region of the pea plastocyanin gene (petE) directed 5–10-fold higher levels of -glucuronidase (GUS) activity than the cauliflower mosaic virus 35S promoter in transgenic tobacco plants, although the levels of GUS mRNA were similar. The sequence (AAAAAUGG) around the translation initiation codon of petE enhanced translation of the GUS mRNA 10-fold compared to translation from the GUS translation initiation codon in transgenic tobacco plants and transfected protoplasts.     

An analysis of tobacco mosaic virus replicative structures synthesized in vitro

Summary The RNA structures synthesized in vitro by a crude enzyme complex from tobacco mosaic virus (TMV)-infected leaves have been analyzed; the major viral-specific products were similar to TMV-replicative form (RF) and-replicative intermediate (RI) in electrophoretic behavior and ribonuclease sensitivity. Synthesis of these RF-like and RI-like structures neither required nor responded to added viral RNA, but did require all four ribonucleotide triphosphates. Enriched radiolabeled RF-like and RI-like RNA fractions were isolated from non-denaturing agarose gels by electroelution and hybridized to a collection of TMV sequences cloned into bacteriophage M13. Enriched RF-RNA hybridized to sequences of both plus and minus polarity, while enriched RI-RNA hybridized only to inserts of minus polarity, indicating only plus strand synthesis in this fraction. Most of the label incorporated into the plus strand of the enriched RF-RNA was found near the 3-end of this strand, while most of the label incorporated into enriched RI-RNA was found several hundred bases from the 5-end of the plus strand.Paper presented at the first International Congress of Plant Molecular Biology (Savannah, GA, 1985).     

PO149, a new member of pollen pectate lyase-like gene family from alfalfa

PO149 is a low-copy-number gene expressed in the late stages of pollen development. The promoter region contains no similarities in DNA sequence to those of other pollen-specific genes, except for a tobacco sequence (AAATGA), which occurs four times in this alfalfa gene and much further upstream than in tobacco. Four distinct TATA boxes were detected in the promoter with the distal and proximal TATA boxes being separated by a spacer of 269 nucleotides. Hairpin loop structures were found in the 5-and 3-untranslated regions of PO149 mRNA. The coding region of PO149 is interrupted by two introns and encodes a putative prepeptide of 450 amino acids with homology to pollen pectate lyase-like proteins and pollen allergens. The coding region also contains sequences characteristic of both a signal peptide and a nuclear localization signal.     

Bell Pepper Mottle Virus, a Distinct Tobamovirus Infecting Pepper

Bell pepper mottle virus (BPeMV) can be distinguished by symptomatology and host range from other tobamoviruses but a reliable identification needs serological tests. The relationships of BPeMV to tobacco mosaic virus (TMV), Odontoglossum ringspot virus (ORSV), tobacco mild green mosaic virus (TMGMV), and pepper mild mottle virus (PMMV) were investigated using precipitin drop tests on slides, immunodiffusion gel tests, double antibody sandwich enzyme-linked immunosorbent assay (ELISA), and indirectELISA using enzyme-linked goat anti-rabbit globulins for the determination of antiserum titers and serological differentiation indices (SDI). Comparisons of SDIs and amino acid composition data demonstrated that BPeMV is a new species of the tobamovirusgroup. BPeMV, ORSV, PMMV, and TMGMV form a cluster within the genus (group) and could be considered as a subgenus of tobamoviruses.     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

Peroxidase-mediated integration of tyramine into xylem cell walls of tobacco leaves

When [2-¹⁴C]tyramine was fed in vivo by petiolar uptake to Nicotiana tabacum Xanthi n.c. leaves partially inoculated with tobacco mosaic virus, radioactivity accumulated in inoculated areas bearing necrotic lesions, mainly in the veins and around the lesions. Light-microscopic autoradiography showed that integration of radioactivity was especially evident in xylem cell walls. This was confirmed in sections of petiole by electron-microscopic autoradiography. Study of the mechanism of insolubilisation of tyramine showed that the amine was integrated in regions in which peroxidase activity could be located cytochemically using 3,3-diaminobenzidine and H₂O₂ as substrates. When sections of petiole were incubated with labelled tyramine and H₂O₂ after fixation in glutaraldehyde, a distribution of radioactivity similar to that obtained after feeding tyramine by petiolar uptake was observed. It is concluded that simple phenols such as tyramine can be integrated in vivo into cell walls because they are oxidised by peroxidases. This result illustrates the difficulty of studying the metabolism of exogenous phenols in plants, especially in lignifying tissues which contain active wall-bound peroxidases.Abbreviations DAB 3,3-diamino-benzidine - TMV tobacco mosaic virus     

Translational efficiency of mRNA in yeast cells is not increased by plant viral leader sequences

Summary Synthetic oligonucleotides encoding the 5-non-translated (leader) sequence of the coat protein mRNA of alfalfa mosaic virus RNA 4 or the leader sequence of tobacco mosaic virus RNA were used to replace the natural leader region of the yeast phosphoglycerate kinase (PGK1) mRNAs and the translational efficiency of the chimeric mRNA was determined in yeast cells. In neither case did we observed a significant increase compared to the translational efficiency shown by the wild-type PGK mRNA, in contrast to the known stimulatory effect of these leader sequences on translation in mammalian, plant and bacterial in-vivo and/or in-vitro systems. The same result was obtained when the translational efficiencies in yeast cells of Escherichia coli -galactosidase mRNAs carrying the PGK or either of the two viral leader sequences were compared. Offprint requests to: H. A. Raué     

Transformation of <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> with different BWYV (Beet western yellows virus) sequences to test for virus resistance

Nicotiana benthamiana plants were transformed by means of Agrobacterium tumefacienscarrying various constructs to test for resistance for BWYV (Beet western yellows virus), the constructs used included either the viral replicase (ORF1/2), one of two smaller sequences involving the 5 and 3 ends (53S and 53AS) of the viral genome of BWYV. According to different criteria such as ELISA, PCR, growth under kanamycin selection at 200 mg l^–1 and phenotype some lines were chosen as candidates to be tested for resistance against BWYV. Five lines of each construct were randomly selected. These lines were analysed by Northern blot for expression of the transgene of interest and/or the nptII gene. Greenhouse resistance tests were performed in 15 transgenic N. benthamiana lines (5 per construct) and in two controls. The transgenic plantlets were transferred to the greenhouse and 20 plants from each line were inoculated with BWYV using the natural vector Myzus persicae, while other 20 were kept as uninoculated control. At 4, 6 and 8 weeks post-infection (wpi) a BWYV ELISA of the inoculated plants was carried out and the height of each plant was measured, while the weight was determined at the end of the experiment. None of the transgenic lines tested showed resistance to the virus.     

Phosphodiesterase activities in transgenic tobacco plants associated with the movement protein of tobacco mosaic virus

Summary Hydrolytic activities of leaf extracts from normal and transgenic plants, with (+ MP) and without (-MP) the movement protein of tobacco mosaic virus, were examined. In the + MP transgenic plants, as compared with non-transgenic and — MP plants, higher hydrolytic activities were found on the following substrates: bis-(nitrophenyl)-phosphate (BPNPP, phosphodiesterase), p-nitrophenyl-(phenyl)-phosphate (PNPPP, nucleotidephosphodiesterase) and thymidine-3-monophosphate p-nitrophenyl ester (T₃MPP; 3nucleotide phosphodiesterase.) The + MP plant lines, as compared with other transgenic plants, exhibited higher nucleotide-phosphodiesterase activity in the soluble as well as in the membrane fraction. Substrate concentration kinetic studies revealed the presence of a nucleotide-phospho-diesterase with a high substrate affinity in the +MP extracts in addition to the enzyme with a relatively low substrate affinity present also in the — MP transgenic plants. This high affinity enzyme could be removed from the soluble fraction by precipitation with anti-MP serum, indicating its possible association with the movement protein.     

Detection of Single Nucleotide Variations in Viral RNA Populations by Primer Extension

A method for identifying and differentiating between two highly similar sequence variants in the cucumber mosaic virus RNA 5 population is described. The technique is based on the use of different primers with 3 terminal mismatches for primer extension analysis. Primers varied in their length, number of 3 mismatches, and sequence context of the mismatch. The results indicate that the size of the primer is critically important to the ability of this method to differentiate between highly similar RNAs. This technique should prove very useful for the genetic analysis of characteristics that are determined by single nucleotide variations in viral RNA genomes.Abbreviations: nt, nucleotide; CMV, cucumber mosaic virus.     

Cell-free translation of exogenous mRNA in extracts from dry pea primary axes

Extracts from the primary axes of dry pea (Pisum sativum L.) seeds are able to perform an initiation-dependent translation of exogenous mRNA. SDS polyacrylamide gel electrophoresis of the products synthesized under direction of alfalfa mosaic virus RNA (AMV-RNA) and tobacco mosaic virus RNA (TMV-RNA) shows that the fidelity of translation in this pea system is at least as high as in a wheat embryo cell-free protein synthesizing system. The endogenous messengers are also efficiently translated in extracts from the primary axes of pea seeds. The direct translation of these messengers in a homologous cell-free system may be of interest for a study of the products coded for by the long-lived messengers present in this plant.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - SDS sodium dodecyl sulphate - mRNP messenger ribonucleoprotein - AMV-RNA alfalfa mosaic virus RNA - TMV-RNA tobacco mosaic virus RNA - ATA aurin tricarboxylic acid - TCA trichloroacetic acid - S.A. specific activity     

cDNA cloning of artichoke mottled crinkle virus RNA and localization and sequencing of the coat protein gene

We report the cDNA cloning of the genomic RNA of artichoke mottled crinkle virus (AMCV), which is a member of Tombusvirus group. AMCV has a monopartite positive sense RNA genome, which is not polyadenylated at the 3 end. The genome size is 4.8 kb.We have localized and sequenced the open reading frame (ORF) encoding the coat protein. Unlike most monopartite positive-strand RNA plant viruses, the ORF is not located near the 3 end, but like other members of the Tombusvirus group, CyRSV (cymbidium ringspot virus), TBSV-cherry (tomato bushy stunt virus cherry strain) and CNV (cucumber necrosis virus) it starts ca. 2.7 kb downstream of the 5 end and stops ca. 1 kb upstream of the 3 end. This ORF predicts a polypeptide chain of 387 amino acids.Comparison of the coat proteins of AMCV, TBSV-BS3, TBSV-cherry and CNV confirms that, within the Tombusvirus group, there exists a high degree of similarity among coat proteins but that this similarity is not uniformly distributed among domains. In particular, the N-terminal region, thought to make contact with the phosphate groups of the viral RNA, and the C-terminal region, considered the most immunogenic portion of the capsid, are found to be the least homologous.     

Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement

Virus-encoded movement protein (MP) mediates cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata. The molecular pathway by which TMV MP interacts with the host cell is largely unknown. To understand this process better, a cell wall-associated protein that specifically binds the viral MP was purified from tobacco leaf cell walls and identified as pectin methylesterase (PME). In addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP showed that its domain was necessary and sufficient for association with PME. Deletion of the PME-binding region resulted in inactivation of TMV cell-to-cell movement.