Encapsidated circular viroid-like satellite RNAs (virusoids) of plants.
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Base Sequence↗
Biomedical subjects
R H Symons
Publications and source records attributed to R H Symons.
Ilarviruses encode a Cucumovirus-like 2b gene that is absent in other genera within the Bromoviridae.
We found that RNA 2 of the four ilarviruses sequenced to date encodes an additional conserved open reading frame (ORF), 2b, that overlaps the 3' end of the previously known ORF, 2a. A novel RNA species of 851 nucleotides was found to accumulate to high levels in plants infected with spinach latent virus (SpLV). Further analysis showed that RNA 4A is a subgenomic RNA of RNA 2 and encodes all of ORF 2b. Moreover, a protein species of the size expected for SpLV ORF 2b was translated in vitro from the RNA 4A-containing virion RNAs. The data support the suggestion that the SpLV 2b protein is translated in vivo. The 2b gene of ilarviruses, which is not encoded by alfamoviruses and bromoviruses, shares several features with the previously reported cucumovirus 2b gene; however, their encoded proteins share no detectable sequence similarities. The evolutionary origin of the 2b gene is discussed.
Amino Acid Sequence↗
Specific RNA self-cleavage in coconut cadang cadang viroid: potential for a role in rolling circle replication.
The rolling circle replication of the small, single-stranded viroid RNAs requires a specific processing reaction to produce monomeric RNAs that are ligated into the final circular form. For avocado sunblotch viroid, peach latent mosaic viroid, and chrysanthemum chlorotic mottle viroid, the hammerhead self-cleavage reaction is considered to provide this processing reaction. We have searched for a similar type of reaction in the 246-nt coconut cadang cadang viroid, the smallest viroid of the 24-member potato spindle tuber viroid (PSTV) group. RNA transcripts prepared from the cloned central or C domain of this viroid self-cleaved specifically after denaturation with methylmercuric hydroxide followed by incubation in the presence of spermidine but in the absence of added magnesium ions. The unique cleavage site was located in the bottom strand of the C domain within a potential hairpin structure that is conserved within members of all three subgroups of the PSTV group of viroids.
Base Sequence↗
Plant pathogenic RNAs and RNA catalysis.
The rolling circle replication of small circular plant pathogenic RNAs requires a processing step to convert multimeric intermediates to monomers which are then circularized. Eleven such RNAs are known so far, two are viroids, one is viroid-like and the remainder are satellite RNAs dependent on a helper virus for replication. The processing step is RNA-catalysed in all cases, at least in vitro. All plus forms of these RNAs self-cleave via the hammerhead structure whereas only eight of the minus RNAs self-cleave, five via the hammerhead structure and three via the hairpin structure. There are about 20 other viroids where the processing mechanism has yet to be determined but they are likely candidates for a new type of self-cleavage reaction which is predicted to be conserved in all these viroids. Hepatitis delta RNA is the only circular pathogenic RNA known to self-cleave in the animal kingdom. It is feasible that more single-stranded circular pathogenic RNAs are waiting to be discovered and these could be prospective for new types of self-cleavage reactions.
Base Sequence↗
Barley yellow dwarf virus-PAV RNA does not have a VPg.
The genus Luteovirus is divided into two subgroups. It has already been shown that a genome-linked protein (VPg) is covalently attached to the 5'-end of the genomic RNA of subgroup II luteoviruses, but whether a VPg is present or absent on the genomic RNA of subgroup I luteoviruses is unknown. In the present work, a simple and sensitive method was developed for the detection of the VPg on virion genomic RNA. This method was used to show that a VPg is not attached to the 5'-end of the RNA genome of barley yellow dwarf virus-PAV which is a member of subgroup I luteoviruses.
Hordeum↗
A high sensitivity RT-PCR assay for the diagnosis of grapevine viroids in field and tissue culture samples.
An RNA extraction procedure, modified from published methods, and a high sensitivity reverse transcription-polymerase chain reaction (RT-PCR) assay have been developed for the detection of the five viroids in grapevines. All five viroids have been found in the 10 different grape varieties tested so far. This assay, specially optimised for viroids in low copy number by careful selection of DNA primers, has been used in conjunction with dot blot hybridization assay for the study of viroids in vines regenerated by shoot apical meristem culture (SAMC) and fragmented shoot apex culture (FSAC). The data indicate a differential reduction of viroids, rather than viroid elimination, in the regenerated vines. Transmission of viroids via grape seeds was also observed.
Fruit↗
In vivo expression of an overlapping gene encoded by the cucumoviruses.
We recently reported the molecular characterization and functional analysis of an overlapping gene 2b encoded by RNA 2 of the Q strain of cucumber mosaic cucumovirus (Q-CMV). We show here that the homologous gene encoded by the V strain of tomato aspermy cucumovirus (V-TAV) and the WAII strain of CMV (WAII-CMV), which is in a different subgroup to Q-CMV, is also expressed in vivo by demonstrating the accumulation of the mRNA (RNA 4A) and its protein in infected plants. Interestingly, RNA 4A of V-TAV is encapsidated in virions as found previously for Q-CMV whereas WAII-CMV contains very little RNA 4A in virions. As the 2b gene is conserved in all 10 cucumoviral species or strains sequenced to date and the 2b gene is expressed for three of these viruses, we conclude that the 2b gene is a common feature of the Cucumovirus genus.
Base Sequence↗
Two novel subgenomic RNAs derived from RNA 3 of tomato aspermy cucumovirus.
Two abundant subgenomic RNAs, designated RNA 3B and RNA 5, were found to be associated with the V strain of tomato aspermy cucumovirus (V-TAV). Sequence determination showed that the 3'-terminal 323 nucleotides (nt) of RNA 3B was identical to RNA 5, whereas its 5'-terminal 163 nt was a direct repeat (one nt difference) of the 5'-half of RNA 5, and that both RNAs are completely homologous to the 3'-terminal untranslated region of TAV RNA 3. TAV RNAs 3B and 5 were also detected in the infection of a pseudorecombinant virus consisting of TAV RNA 3 and RNAs 1 and 2 from cucumber mosaic virus. Furthermore, only RNA 5, not RNA 3B, was detected in a TAV mutant in which one of the repeats was deleted from RNA 3. These genetic studies clearly show that both RNA species are derived from TAV RNA 3. However, in contrast to TAV RNAs 4 and 4A, which encode coat protein and 2b protein, respectively, RNAs 3B and 5 represent a novel class of subgenomic RNAs from TAV that do not function as mRNAs. Possible functional roles for such a class of viral subgenomic RNAs are discussed.
Base Sequence↗
Plasmid vector for cloning infectious cDNAs from plant RNA viruses: high infectivity of cDNA clones of tomato aspermy cucumovirus.
An improved version of the previously obtained cloning vector pCass was constructed by partially duplicating the 35S promoter used to drive the transient transcription of cloned viral cDNAs. Full-length cDNAs of the three genomic RNAs of tomato aspermy cucumovirus (TAV) cloned in this improved pCass (designated pCass2) gave a 3-fold higher infectivity in two plant species tested than the same cDNAs cloned in pCass1 with only a single 35S promoter. Host range, symptoms, morphology of viral particles and viral progeny RNAs induced by these sets of infectious cDNA clones analysed were identical to those induced by the wild-type virus. A mutant of genomic TAV RNA 3 containing a 163 nt deletion in the 3' untranslated region was stably maintained in the progeny RNAs, indicating that these cDNA clones may facilitate a study of virus function. This is the first report of infectious cDNA clones of TAV as well as of infectious cDNA clones with a duplicated 35S promoter of CaMV.
Cloning, Molecular↗
An interspecies hybrid RNA virus is significantly more virulent than either parental virus.
Cucumber mosaic cucumovirus (CMV) infects a very wide range of plant species (>1000 species). We recently demonstrated that a previously undescribed gene (2b) encoded by RNA 2 of the tripartite RNA genome of CMV is required for systemic virus spread and disease induction in its hosts. Herein we report that when this CMV gene is replaced by its homologue from tomato aspermy cucumovirus (TAV), the resultant hybrid virus is significantly more virulent, induces earlier onset of systemic symptoms, and accumulates to a higher level in seven host species from three families than either of the parents. Our results indicate that CMV and the TAV 2b protein interact synergistically despite the fact that no synergism occurs in double infections with the two parental viruses. To our knowledge, this is the first example of an interspecific hybrid made from plant or animal RNA viruses that is more efficient in systemic infection of a number of hosts than the naturally occurring parents. As CMV and the hybrid virus accumulated to a similar level in the infected tobacco protoplasts, the observed synergistic responses most likely resulted from an increased efficacy of the hybrid virus in systemic spread in host plants provided by the TAV 2b protein. The relevance of our finding to the application of pathogen-derived resistance is discussed.
Base Sequence↗
Sequence analysis of the second largest subunit of tomato RNA polymerase II.
We have cloned and sequenced the cDNA encoding the open reading frame of the mRNA of the second largest subunit of RNA polymerase II, or RPB2, of tomato. The mRNA is transcribed from a single-copy gene in the tomato genome and the transcript size of the gene was measured as 4.2 kb by northern analysis. From the deduced amino acid sequence of 1191 residues, a protein of M r 135 000 with an isoelectric point of pH 7.9 was predicted. Alignment of the tomato RPB2 protein sequence with those of the homologous subunits in Arabidopsis, man, Drosophila and yeast showed considerable sequence identity.
Amino Acid Sequence↗
A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement.
We recently identified a new cucumovirus-specific gene (2b) which is encoded by RNA 2 of the cucumber mosaic cucumovirus (CMV) tripartite RNA genome and whose coding sequence overlaps the C-terminal 69 codons of ORF 2a encoding the RNA polymerase protein. We have now found that although a CMV mutant lacking ORF 2b accumulated in the inoculated cotyledons of cucumber plants, it was unable to spread systemically, demonstrating involvement of 2b in long distance movement. The same mutant infected tobacco systemically with a much reduced virulence and delayed appearance of symptoms, indicating that 2b may contribute to long distance movement in this host. Deletion of the overlapping C-terminal part of ORF 2a did not change infectivity of the mutant in either host species, ruling out 2a mutation as the reason for the change of phenotype. Further infectivity studies with mutants containing partial deletions in ORF 2b further supported the conclusion that 2b encodes a host-specific long distance movement function. Sequence analysis revealed that 2b may represent a novel naturally occurring hybrid gene important to the evolutionary formation of the cucumovirus group and that it could provide a genetic basis for the wide host range of these viruses.
Base Sequence↗
Efficient infection from cDNA clones of cucumber mosaic cucumovirus RNAs in a new plasmid vector.
Full-length cucumber mosaic cucumovirus (CMV) cDNAs were cloned into a new plasmid vector containing a modified plant virus promoter designed to transcribe the inserted sequence from its first nucleotide. cDNA copies of CMV strain Q (Q-CMV) genomic RNAs 1, 2 and 3 cloned into this vector were infectious when inoculated together, producing symptoms indistinguishable from those caused by wildtype Q-CMV infection. The infectivity of the clones could be substantially increased by excision of the viral insert together with the transcriptional promoter and terminator prior to inoculation. A diagnostic but silent mutation was introduced into the RNA 2 cDNA and found to be stably maintained in viral infection, allowing distinction of the recombinant virus from native contaminants. The infectious cDNA clones supported the replication of CMV satellite RNA when co-inoculated with biologically active Q-CMV satellite RNA transcripts. Using the infectious cDNAs described, it was found that a newly-identified overlapping gene (2b) encoded by Q-CMV RNA 2 was not essential for either systemic viral infection of Nicotiana glutinosa or replication of the satellite RNAs.
Base Sequence↗
Probing the hammerhead ribozyme structure with ribonucleases.
Susceptibility to RNase digestion has been used to probe the conformation of the hammerhead ribozyme structure prepared from chemically synthesised RNAs. Less than about 1.5% of the total sample was digested to obtain a profile of RNase digestion sites. The observed digestion profiles confirmed the predicted base-paired secondary structure for the hammerhead. Digestion profiles of both cis and trans hammerhead structures were nearly identical which indicated that the structural interactions leading to self-cleavage were similar for both systems. Furthermore, the presence or absence of Mg2+ did not affect the RNase digestion profiles, thus indicating that Mg2+ did not modify the hammerhead structure significantly to induce self-cleavage. The base-paired stems I and II in the hammerhead structure were stable whereas stem III, which was susceptible to digestion, appeared to be an unstable region. The single strand domains separating the stems were susceptible to digestion with the exception of sites adjacent to guanosines; GL2.1 in the stem II loop and G12 in the conserved GAAAC sequence, which separates stems II and III. The absence of digestion at GL2.1 in the stem II hairpin loop of the hammerhead complex was maintained in uncomplexed ribozyme and in short oligonucleotides containing only the stem II hairpin region. In contrast, the G12 site became susceptible when the ribozyme was not complexed with its substrate. Overall the results are consistent with the role of Mg2+ in the hammerhead self-cleavage reaction being catalytic and not structural.
Base Composition↗
New overlapping gene encoded by the cucumber mosaic virus genome.
Cucumber mosaic virus (CMV) contains three genomic RNAs (RNAs 1, 2, and 3) and a subgenomic RNA (RNA 4), a shared feature of the Bromoviridae family which includes cucumoviruses, bromoviruses, alfalfa mosaic virus, and ilarviruses. We describe in this paper the molecular characterization of a novel subgenomic RNA of the Q strain of CMV (Q-CMV), RNA 4A, which was first reported in 1973 (K. W. C. Peden and R. H. Symons, Virology 53, 487-492, 1973). RNA 4A is 682 nucleotides and is identical in sequence to the 3'-terminal 682 nucleotides of RNA 2. RNA 4A encodes a small open reading frame (ORF) of 100 codons, which, in RNA 2, overlaps the C-terminal portion of the major 2a gene; thus it is likely that RNA 4A functions as the mRNA for the in vivo expression of the ORF, called ORF 2b. Polyclonal antibodies raised against a 2b fusion protein expressed in Escherichia coli specifically detected the 2b gene product in Q-CMV-infected cucumber plants by Western immunoblotting. Examination of published viral RNA sequences revealed the conservation of ORF 2b in all of the four other cucumoviruses sequenced to date; however, it is absent from the rest of the Bromoviridae. We suggest that the proposed ORF 2b may be expressed in other cucumoviruses, most likely via 4A-like subgenomic RNAs, and that the predicted gene product may have a unique functional role in the infection process of cucumoviruses.
Amino Acid Sequence↗
Soybean dwarf luteovirus contains the third variant genome type in the luteovirus group.
Complementary DNAs covering the entire RNA genome of soybean dwarf luteovirus (SDV) were cloned and sequenced. Computer analysis of the 5861 nucleotide sequence revealed five major open reading frames (ORFs) possessing conservation of sequence and organisation with known luteovirus sequences. Comparative analyses of the genome structure show that SDV shares sequence homology and features of gene organisation with barley yellow dwarf virus (PAV isolate) in the 5' half of the genome, yet is more closely related to potato leafroll virus in its 3' coding regions. In addition, SDV differs from other known luteoviruses in possessing an exceptionally long 3' terminal sequence with no apparent coding capacity. We conclude from these data that the SDV genome represents a third variant genome type in the luteovirus group.
Amino Acid Sequence↗
Infectivity of linear monomeric transcripts of citrus exocortis viroid: terminal sequence requirements for processing.
Infectivity of linear monomeric viroid RNA transcripts is known to be dependent on the site of cloning of the viroid and the viroid and vector sequences at the termini of the RNA transcripts. We report here the further characterization of cloning site and sequence requirements using 11 different monomeric cDNA constructs of citrus exocortis viroid; these differed from one another by the site of cloning and by nonviroid residues adjacent to the 5' and 3' termini of the viroid sequence in the RNA transcript. The infectivity of these transcripts was assayed by inoculation onto tomato seedlings; all infectious transcripts were wild type, indicating accurate processing. The basic requirement for infectivity appeared to be the ability of the RNA transcripts to form a short double-stranded region of viroid and vector sequences at the junction of the two termini. The site of cloning of the viroid RNA used for cloning was not important as cDNA clones prepared at seven sites provided infectious transcripts.
Base Composition↗
A stable 463 nucleotide variant of citrus exocortis viroid produced by terminal repeats.
An unusual variant of citrus exocortis viroid (CEV) was detected when an inoculum source from Gynura aurantiaca D.C. was used to infect a hybrid tomato (Lycopersicon esculentum Mill. x L. peruvianum). The 92 nucleotide larger variant, CEV D-92, which displayed the characteristic circular and linear viroid structural forms, contained two repeated sequences spanning the V and T2 domains. A dramatic moderation of symptom expression in Gynura accompanied the incorporation of these repeated sequences. A comparison of the sequence and structure of CEV D-92 with coconut cadang-cadang viroid revealed similarities in the regions generating the naturally occurring terminal repeats suggesting a possible preferred site for RNA recombination between viroids.
Base Sequence↗