Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Viroids”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources
PubMed1982

Stiffness of viroids and viroid-like RNA in solution.

The sedimentation coefficients of the potato spindle tuber viroid, four viroid-like RNAs from cadang-cadang-disease, circular RNA from velvet tobacco mottle virus, circular RNA from Solanum nodiflorum mottle virus and double stranded RNA5 from cucumber mosaic virus were measured in the analytical ultracentrifuge. The numbers of nucleotides of the RNA species varied between 246 and 670. The hydrodynamic models of rigid rods and flexible cylinders were applied for the interpretation of the sedimentation coefficients. Double-stranded RNA5 from cucumber mosaic virus with 335 basepairs fits the model of a rigid rod with an hydrated diameter of 29 A. Potato spindle tuber viroid and the four viroid-like RNA species of cadang-cadang-disease form a homologous series of flexible cylinders with a Kuhn's statistical length lambda-1 of 600 A. The circular RNA from the two viruses mentioned above are more flexibel than the viroids and viroid-like RNAs. The hydrodynamic interpretation is in accordance with thermodynamic data and secondary structure models. In two of the RNAs from cadang-cadang, cruciform structures would also be possible on the basis of the nucleotide sequence. The hydrodynamic data, however, favour clearly the extended structure over the cruciform.

Genes, Viral↗
PubMed1985

Infectivity and in vitro mutagenesis of monomeric cDNA clones of citrus exocortis viroid indicates the site of processing of viroid precursors.

Monomeric cDNA clones of citrus exocortis viroid (CEV) were constructed in the plasmid vector pSP6-4 and the infectivity of the clones plus in vitro-synthesized RNA transcripts determined by inoculation onto tomato seedlings. Infectivity was dependent on the site of the viroid molecule used for cloning and the orientation of the cDNA insert. Only the plus BamHI cDNA clone was infectious and produced progeny viroid with wild-type sequence at the region corresponding to the BamHI cloning site. Infectivity correlated with the terminal repetition of 11 nucleotides of viroid sequence, 5'GGATCCCCGGG 3', in the vector adjacent to the insert. The 11-nucleotide sequence lies within the highly conserved central region of viroids. Site-directed mutagenesis of a single nucleotide in the repeat at the 5'-end of the CEV insert to 5' GGATCCCC(T,A)GG 3' gave two point mutants. The two mutant CEV inserts, when excised from the vector, were not infectious. However, plasmid DNA and RNA transcripts from non-excised mutant CEV inserts were infectious. The progeny of one of these clones was examined and contained wild-type sequence. It was concluded that in vivo processing of longer-than-unit-length CEV occurs at one of three adjacent sites in the 11 nucleotide sequence and that the G nucleotide at position 97 is important for viroid replication.

Base Sequence↗
PubMed1986

Structure of viroid replicative intermediates: physico-chemical studies on SP6 transcripts of cloned oligomeric potato spindle tuber viroid.

The structure and structural transitions of transcripts of cloned oligomeric viroid were studied in physico-chemical experiments and stability calculations. Transcripts of (+) and (-) polarity, from unit up to sixfold length, were synthesized from DNA clones of the potato spindle tuber viroid (PSTV) with the SP6 transcription system. Their structural properties were investigated by optical denaturation curves, high performance liquid chromatography (HPLC), electron microscopy, sedimentation-diffusion equilibrium and velocity sedimentation. Secondary structures of the RNAs and theoretical denaturation curves were calculated using an energy optimization program. The secondary structure of lowest free energy for unit length and oligomeric transcripts is a rod-like structure similar to that of the mature circular viroids. When this structure is used as a model for calculations, there is a large degree of agreement between the theoretical and the experimental denaturation curves. At high temperatures, however, (+) strand transcripts exhibited a transition which was more stable than expected from the calculations or than was known from curves of mature viroids. This transition arises from a rearrangement of the central conserved region of viroids to a helical region of 28 stable base pairs either intermolecularly leading to bimolecular complexes, or intramolecularly giving rise to a branched secondary structure. The rearrangement could be detected by electron microscopy, HPLC, and analytical ultracentrifugation. The helical region serves to divide up the oligomeric (+) strand into structural units which may be recognized by cleavage and ligation enzymes which process the oligomeric intermediates to circular mature viroids.

Microscopy, Electron↗
PubMed1992

Pear blister canker viroid is a member of the apple scar skin subgroup (apscaviroids) and also has sequence homology with viroids from other subgroups.

The sequence of pear blister canker viroid (PBCVd), the putative causal agent of pear blister canker (PBC) disease, has been determined. PBCVd consists of a single-stranded circular RNA of 315 nucleotide residues which assumes a branched conformation when it is folded in the model of lowest free energy. PBCVd has highest sequence similarity with grapevine 1B viroid (52.4%), but also contains sequences related to regions present in viroids that belong to different subgroups, suggesting that PBCVd could have developed from RNA recombination between viroids replicating in a common host plant. PBCVd contains almost the entire central sequence which is conserved in the members of the apple scar skin subgroup (apscaviroids) as well as a conserved sequence located in the left-terminal region of apscaviroids and pospiviroids (whose type member is potato spindle tuber viroid). A consensus phylogenetic tree has been obtained in which PBCVd and other viroids previously classified as apscaviroids appear closely related, allowing consideration of PBCVd as a new member of this subgroup.

Base Sequence↗
PubMed1996

Variation of viroid profiles in individual grapevine plants: novel grapevine yellow speckle viroid 1 mutants show alterations of hairpin I.

This is the first report which gives a general survey about viroid variant composition in a vineyard and within single plants. A German vineyard with 20-year-old grapevines (Vitis vinifera) of the cultivars 'Bacchus' and 'Kerner' was analysed for viroid infections. Only grapevine yellow speckle viroid 1 (GYSVd1) and the grapevine isolate of hop stunt viroid (HSVdg) were detected. Both viroids occur in several sequence variations. Eighteen novel GYSVd1 variants and two previously published HSVdg main variants with six new minor variants were found. They were randomly spread in the vineyard. The distribution of GYSVd1 and HSVdg main variants and their accompanying subvariants differed even in neighbouring plants. We conclude that these individual viroid variant profiles are the result of 20 years of independent evolution, i.e. mutation and selection, in each single plant. Four of the nine GYSVd1 main variants were mutated in the inverted repeats bordering the central conserved region. These base substitutions decreased the thermodynamic stability of a metastable structure called hairpin I.

Base Sequence↗
PubMed1998

Regulation of pathogenicity in hop stunt viroid-related group II citrus viroids.

Nucleotide sequences were determined for two hop stunt viroid-related Group II citrus viroids characterized as either a cachexia disease non-pathogenic variant (CVd-IIa) or a pathogenic variant (CVd-IIb). Sequence identity between the two variants of 95.6% indicated a conserved genome with the principal region of nucleotide difference clustered in the variable (V) domain. Full-length viroid RT-PCR cDNA products were cloned into plasmid SP72. Viroid cDNA clones as well as derived RNA transcripts were transmissible to citron (Citrus medica L.) and Luffa aegyptiaca Mill. To determine the locus of cachexia pathogenicity as well as symptom expression in Luffa, chimeric viroid cDNA clones were constructed from segments of either the left terminal, pathogenic and conserved (T1-P-C) domains or the conserved, variable and right terminal (C-V-T2) domains of CVd-IIa or CVd-IIb in reciprocal exchanges. Symptoms induced by the various chimeric constructs on the two bioassay hosts reflected the differential response observed with CVd-IIa and -IIb. Constructs with the C-V-T2 domains region from clone-IIa induced severe symptoms on Luffa typical of CVd-IIa, but were non-symptomatic on mandarin as a bioassay host for the cachexia disease. Constructs with the same region (C-V-T2) from the clone-IIb genome induced only mild symptoms on Luffa, but produced a severe reaction on mandarin, as observed for CVd-IIb. Specific site-directed mutations were introduced into the V domain of the CVd-IIa clone to construct viroid cDNA clones with either partial or complete conversions to the CVd-IIb sequence. With the introduction of six site-specific changes into the V domain of the clone-IIa genome, cachexia pathogenicity was acquired as well as a moderation of severe symptoms on Luffa.

Base Sequence↗
PubMed1998

Genomic structure of three phenotypically different isolates of peach latent mosaic viroid: implications of the existence of constraints limiting the heterogeneity of viroid quasispecies.

The peach latent mosaic viroid (PLMVd) is used to study the interactions between a viroid containing hammerhead ribozymes and its natural host, peach. To gain insight into the molecular basis of the phenotypic effects observed upon viroid infection, sequence variants from three PLMVd isolates that differ in symptom expression on the peach indicator GF-305 have been characterized. Analysis of the primary structures of a total of 29 different sequence variants derived from a severe and two latent isolates has revealed a large number of polymorphic positions in the viroid molecule. The variability pattern indicates that preservation of the stability of both hammerhead structures and conservation of a branched secondary structure of the viroid molecule may be factors limiting sequence heterogeneity in PLMVd. Moreover, compensatory mutations in two hairpin loops of the proposed secondary structure, suggesting that a pseudoknot-like interaction may exist between them, have also been observed. Phylogenetic analysis has allowed the allocation of PLMVd molecules into three major groups. This clustering does not strictly correlate with the source isolate from which the variants were obtained, providing insights into the complex mixture of molecules which make up each isolate. Bioassays of individual PLMVd sequence variants on GF-305 peach seedlings have shown that the biological properties of the PLMVd isolates may be correlated with both the complexity of their viroid populations and the presence of specific sequence variants.

Base Sequence↗
PubMed1992

Transcripts of the viroid central conserved region contain the local tertiary structural element found in full-length viroid.

The viroid central conserved region (CCR) is highly conserved among different viroids and is thought to be involved in viroid replication. A novel tertiary structure occurs in the CCR of native circular potato spindle tuber RNAs. To permit more detailed studies of this structural element, a small RNA oligonucleotide containing the CCR of the viroid genome was synthesized. The tertiary structure of these CCR transcripts was examined by UV-crosslinking of the RNA, followed by mapping of the crosslink using limited alkaline digestion and classical RNA secondary analysis. The CCR transcript was found to undergo UV-crosslinking between the same two bases as in full-length viroid, indicating that the tertiary structure is the same and that the CCR transcript will be useful for the affinity purification of host components.

Base Sequence↗
PubMed1995

The termini of a new citrus viroid contain duplications of the central conserved regions from two viroid groups.

A citrus viroid associated with dwarfing, CVdIIIA, has been sequenced and its 294 nucleotide residues can be arranged to form the typical rod-like secondary structure of other viroids with 71% of nucleotides base-paired. CVdIIIA has greatest sequence similarity with apple scar skin viroid (ASSVd; 69%) and has the central sequence which is conserved in the ASSVd group. CVdIIIA is the smallest member of the ASSVd group but contains the terminal conserved region shared by all viroids over 300 nucleotides. The two ends of CVdIIIA are highly unusual in that each end appears to be derived from the conserved central core region of a different viroid group.

Base Sequence↗
PubMed1989

Infectivity of chimeric viroid transcripts reveals the presence of alternative processing sites in potato spindle tuber viroid.

In an investigation of viroid replication and pathogenesis, we have assessed the effect of sequence duplication of the upper central conserved region (CCR) of the molecule on the infectivity of RNAs transcribed in vitro from partial dimers of wild-type and mutant viroid cDNAs. In one set of experiments, the relative infectivities of one monomeric potato spindle tuber viroid (PSTV) and five oligomeric SP6 transcripts [PSTV or PSTV-TASV (tomato apical stunt viroid) chimeras] were compared. With one exception, the extent of sequence duplication in the CCR, and thus the length of the so-called palindrome, does correlate with an increase in specific infectivity. In a second set of experiments, in vitro generated site-specific mutations in cloned PSTV were used as markers to determine if a cleavage/ligation at sites other than the palindrome could generate infectious molecules in vivo. The creation of a novel PSTV-TPMV (tomato planta macho viroid) chimera in these experiments provides evidence that multimeric RNAs can be processed at sites other than the CCR to yield monomeric progeny.

Base Sequence↗
PubMed1993

Mechanism of viroid pathogenesis: differential activation of the interferon-induced, double-stranded RNA-activated, M(r) 68,000 protein kinase by viroid strains of varying pathogenicity.

Purified potato spindle tuber viroid (PSTVd) was added to an in vitro assay system containing purified interferon-induced, dsRNA-activated protein kinase (P68). Viroid RNA activated (phosphorylated) the enzyme, although with less efficiency than did the synthetic, perfectly matched poly I-poly C. In binding experiments, RNA transcripts of the intermediate strain of PSTVd were shown to specifically bind to a P68-antibody complex. Activation of the enzyme by a strain of PSTVd that results in severe symptoms in infected tomato plants was at least ten-fold that by the mild strain. Activation by a strain that results in intermediate symptoms was quantitatively similar to activation by the severe strain. To our knowledge, this is the first demonstration of a differential effect of viroid strains inducing different levels of pathology on any biochemical or metabolic system investigated. This differential effect suggests that activation of a plant enzyme homologous to mammalian P68 protein kinase may represent the triggering event in viroid pathogenesis. Differential activation of P68 is surprising, because the primary structures of the mild and severe PSTVd strains analyzed differ by only a two-nucleotide inversion (UUC-->CUU) in the lower portion of the 'pathogenicity' region of the molecules. This change, according to thermodynamic calculations, should have only a minor effect on the secondary structure of the viroid molecule. Binding assays indicated that PSTVd specifically binds to P68.

Animals↗
PubMedDate not supplied

32P- and biotin-labelled in vitro transcribed cRNA probes for the detection of potato spindle tuber viroid and chrysanthemum stunt viroid.

Replacing nick-translated DNA probes by in vitro transcribed complementary RNA (cRNA) probes considerably increased the sensitivity of dot-blot detection tests of potato spindle tuber viroid and chrysanthemum stunt viroid. As compared to the limit of detection of 5-10 pg of viroid obtained with 32P-labelled DNA probes, cRNA probes allow the detection of less than 1 pg of pure viroid. When labelled with biotin by incorporation of biotin-labelled ribonucleotides, the cRNA probes have a limit of detection of approximately 5 pg of purified viroid.

Biotin↗
PubMed1984

Viroid replication: equilibrium association constant and comparative activity measurements for the viroid-polymerase interaction.

The binding and replication of purified potato spindle tuber viroid (PSTV) by DNA-dependent RNA polymerase II from wheat germ was studied in analytical ultracentrifugation experiments and in vitro transcription assays. The equilibrium association constant for the viroid-polymerase interaction is 1.9 X 10(7) M-1. Both ultraviolet and fluorescent monitoring during the sedimentation experiments showed two distinguishable viroid-polymerase complexes. These are interpreted as resulting from a 1:1 and 2:1 enzyme-to-viroid binding stoichiometry. A265/A280 ratios across the sedimenting boundaries, the sedimentation velocity of the complexes, as well as electron microscopic data support this interpretation. The role of viroid secondary structure in enzyme binding and polymerization is discussed in the light of these results and compared with binding and polymerization data for virusoid RNA, single- and double-stranded RNA, and double-stranded DNA.

Microscopy, Electron↗
PubMed1987

The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid.

The primary structure of a grapevine viroid (GVs) isolated in Spain was determined. The sequence consisted of 369 nucleotide residues forming a circular molecule. GVs presented extensive homology with viroids of the potato spindle tuber viroid (PSTV) group, that was specially high in the case of citrus exocortis viroid (CEV) both with variants found in isolates inducing severe (92% with CEV-A) and mild (89% with CEV-DE26) symptoms on tomato. The secondary structure proposed for GVs showed that the changes in the sequence in relation to CEV-A generated modifications of the secondary structure particularly important in the left terminal (Tl), variable (V) and pathogenesis (P) viroid domains that have been postulated. Nevertheless it was noted in GVs a central core in the P domain that is conserved in the class A sequence variants characteristic of severe isolates, but not in the class B ones found in mild isolates of CEV. These observations indicate that GVs should be considered as a severe isolate of CEV from grapevine (CEV-g), a suggestion that correlates with the biological properties of CEV-g both in tomato and in Gynura aurantiaca. The presence of this central core in the P domain seems to characterize all the variants of CEV inducing severe symptoms in tomato.

Citrus↗
PubMed1996

Compilation and analysis of viroid and viroid-like RNA sequences.

We have created a catalogue comprising all viroid and viroid-like RNA sequences which to our knowledge have been either published or were available from on-line sequence libraries as of October 1, 1995. In the development of this catalogue nomenclature ambiguities were removed, the likely ancestral sequence of most species was determined and the most stable secondary structures of these sequences were predicted using the MulFold package. Only viroids of PSTVd-type possessed a rod-like secondary structure, while most other viroids adopted branched secondary structures. Several viroids have predicted secondary structures that include either a Y or cruciform structure reminiscent of the tRNA-like end of virus genomes at an extremity. However, it remains unknown whether or not these predicted structures are adopted in solution, and if they serve a particular function in vivo. Additional information such as the position of the self-catalytic domains are included in the catalogue. An analysis of the data compilated in the catalogue is included. The catalogue will be available on the world wide web (http://www.callistro.si.usherb.ca/jpperra), on computer disk and in printed form. It should provide an excellent reference point for further studies.

Base Sequence↗
PubMed2004

Viroid-induced RNA silencing of GFP-viroid fusion transgenes does not induce extensive spreading of methylation or transitive silencing.

Viroid infection is associated with the production of short interfering RNAs (siRNAs), a hallmark of post-transcriptional gene silencing (PTGS). However, viroid RNAs autonomously replicating in the nucleus have not been shown to trigger the degradation of homologous RNA in the cytoplasm. To investigate the potential of viroids for the induction of gene silencing, non-infectious fragments of potato spindle tuber viroid (PSTVd) cDNA were transcriptionally fused to the 3' end of the green fluorescent protein (GFP)-coding region. Introduction of such constructs into tobacco plants resulted in stable transgene expression. Upon PSTVd infection, transgene expression was suppressed and partial de novo methylation of the transgene was observed. PSTVd-specific siRNA was detected but none was found corresponding to the gfp gene. Methylation was restricted almost entirely to the PSTVd-specific part of the transgene. Neither a gfp transgene construct lacking viroid-specific elements was silenced nor was de novo methylation detected, when it was introduced into the genetic background of the PSTVd-infected plant lines containing silenced GFP:PSTVd transgenes. The absence of gfp-specific siRNAs and of significant methylation within the gfp-coding region demonstrated that neither silencing nor DNA methylation spread from the initiator region into adjacent 5' regions.

Base Sequence↗
PubMed1999

Subcellular localization and rolling circle replication of peach latent mosaic viroid: hallmarks of group A viroids.

We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. No multimeric conformer was detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed.

Fruit↗
PubMed1994

An infectious viroid RNA replicon evolved from an in vitro-generated non-infectious viroid deletion mutant via a complementary deletion in vivo.

The 359 nucleotides (nt) long potato spindle tuber prototype viroid (PSTVd) is sensitive to experimentally introduced mutations as the substitution or deletion of a single nucleotide usually abolishes its infectivity, although certain sequence alterations are tolerated. This is illustrated by the fact that viroid progeny can evolve in planta upon inoculation with substitution mutants generated in vitro, and by the existence of genetically stable 356-360 nt long PSTVd field isolates. However, to date, no viable in vitro-generated deletion mutant of PSTVd has been reported. We have now found a 341 nt long infectious PSTVd RNA replicon that evolved in agrotransformed plants transformed with the dimeric form of an in vitro-deleted, non-infectious 350 bp long PSTVd cDNA unit by an additional complementary deletion of 9 nt in vivo. This is the first report that the deletion-abolished infectivity of a viroid is restored by an additional deletion that concurrently restabilized its perturbed secondary structure by abandoning an internal segment of the rod-like molecule. The fact that approximately 5% of the total PSTVd RNA genome was deleted demonstrates that the maintenance of this viroid-specific rod-like structure is not only essential for nuclease protection but also for the infectivity, i.e. transmissibility, replicability, processibility and pathogenicity of these minimal infectious agents.

Base Sequence↗