Viroid RNA Research Articles

Intra- and Intermolecular Nonenzymatic Ligations Occur within Transcripts Derived from the Peach Latent Mosaic Viroid

We report here the nonenzymatic self-ligation of transcripts corresponding to the peach latent mosaic viroid (PLMVd). This is the first description of this process with viroid sequences, although it has been reported to occur with human hepatitis delta virus RNA. Self-ligation occurs when the 5′-hydroxyl and the 2′,3′-cyclic phosphate termini produced by the hammerhead self-cleavage of the viroid RNA are juxtaposed by the viroid rod-like structure, and a phosphodiester bond is formed between the two following hydrolysis of the cyclic phosphate. Unit-length transcripts undergo intramolecular folding, and their subsequent self-ligation produces circular molecules. The self-ligation observed in vitro may contribute to PLMVd circularization during rolling circle replication; however, this does not exclude the possibility that a host RNA ligase catalyzes the ligation steps in vivo . Like self-cleavage, self-ligation is probably an ancestral reaction, and the enzyme-catalyzed ligation most likely evolved from this primitive mechanism, Furthermore, the intermolecular self-ligation of annealed transcripts derived from PLMVd is demonstrated, suggesting a possible mechanism for sequence reassortment in viroids.

Identification of a retroviroid-like element from plants.

The biological nature of carnation small viroid-like RNA (CarSV RNA), a 275-nt circular molecule with self-cleaving hammerhead structures in its strands of both polarities, was investigated. The lack of infectivity observed in a series of transmission assays in carnation indicates that CarSV RNA, in spite of sharing structural similarities with viroid and viroid-like satellite RNAs from plants, does not belong to either of these two groups. Additional evidence in this direction comes from the observation that CarSV RNA also exists in carnation plants as DNA tandem repeats. In this respect, CarSV RNA is similar to a small transcript of a tandemly repeated DNA sequence of the newt genome. Moreover, CarSV and newt RNAs have similarities in their sequences as well as in some characteristics of their corresponding hammerhead structures. Further analyses have revealed that CarSV DNA is found directly fused to DNA sequences of carnation etched ring caulimovirus, a pararetrovirus, most likely in the form of an extrachromosomal element. The properties of the CarSV RNA/DNA system are those of a retroviroid-like element having some features in common with viroid and viroid-like satellite RNAs from plants and others with the newt transcript.

Structural sites specific to citrus viroid groups

Synthesis of cDNA probes by random-priming of a viroid template displays the unusual property of specificity to all members included within a single citrus viroid Group. The specificity of hybridization reactions was influenced by the structural conformation of the viroid RNA template, reaction conditions for reverse transcription and hybridization protocols. Mapping the loci for probe transcription from the CEVd, CVd-IIb, and CVd-IV genomes suggests that a similar structured conformation may be responsible for group specificity. A stem-loop configuration in the viroid template and hybridization target sites can be proposed to be responsible for the availability of the group-specific sequences.

Agrobacterium-mediated transfer of geminiviruses to plant tissues.

AbstractAgrobacterium-mediated transfer of cloned DNA copies of infectious viral genomes to plant tissues has been termed “agroinoculation” or “agroinfection” (for review, see ref. 1). The technique has been used successfully for a wide variety of virus groups, including the caulimoviruses and badnaviruses (genomes of double-stranded [ds] DNA) (2–4) the geminiviruses (ssDNA genomes) (5–7), the phloem-restricted luteoviruses (ssRNA genomes) (8), as well as for viroids (ssRNA) (9). For viruses with RNA genomes and viroids, cloned cDNA copies of the viral genome are required. Agroinfection of viruses (or viroids) with circular genomes requires the insertion of tandem multimeric copies of the genome between the T-DNA borders of an Agrobacterium binary vector, but for RNA viruses with linear genomes a cDNA copy of the genome must be inserted between a promoter (usually, the cauliflower mosaic virus 35S promoter [CaMV 35S]) and a transcription termination signal (e.g., the nopaline synthase terminator) (8).KeywordsBinary VectorCoat Protein GeneTomato Yellow Leaf Curl VirusAgrobacterium StrainMaize Streak VirusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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.

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.

Multimeric non-radioactive cRNA probes improve detection of potato spindle tuber viroid (PSTVd)

Experimental data showed that multimeric, complementary RNA (cRNA) probes, labelled with non-radioactive digoxigenin (DIG), improved sensitivity of detection of the potato spindle tuber viroid (PSTVd) RNA by 2- to 30-fold as compared with corresponding multimeric cDNA probes. The degree of PSTVd detectability improvement depended upon the type of alkaline phosphatase substrate (colorimetric vs. chemiluminescent) used. Use of hexameric DIG-labelled cRNA probes in combination with chemiluminescent (Lumi-Phos 530) substrate resulted in detection of 0.48 pg of PSTVd RNA. The size of the synthesized cRNA probes corresponded to the size of the respective PSTVd cDNA templates. Interestingly, there was no relationship between the size of the synthesized, DIG-labelled DNA probes and that of the PSTVd cDNA template. This type of anomaly was not observed with other plant viral cDNA templates. Monomeric or multimeric cDNA probes detected both a mild and a severe PSTVd strain in viroid-infected potato leaf extracts diluted 1024 to 2048 times. In comparison, cRNA probes exhibited a much greater dilution end point; PSTVd RNA was detectable in viroid-infected potato leaf tissue diluted up to 16 384 times. Comparable levels of PSTVd sensitivity of detection were obtained with viroid-infected potato tuber tissue.

Gel dependence of electrophoretic mobilities of double-stranded and viroid RNA and estimation of the contour length of a viroid by gel electrophoresis.

Double-stranded (ds) RNA normally exhibits a lower electrophoretic mobility than dsDNA having the same number of base pairs. This has been attributed to its net charge density that is lower than that of B-form DNA. But we show here that dsRNA runs faster than corresponding DNA in gels containing either > or = 2.5% agarose or > or = 8% acrylamide with high crosslinking (19:1 acrylamide:N,N'-methylenebisacrylamide). However, the relative mobility of dsRNA as compared with DNA, extrapolated to 0% gel (0%T), remains constant (0.90 +/- 0.03) in all systems, in support of the charge density hypothesis. In comparison to dsRNA standards, the potato spindle tuber viroid, a small approximately 70% base-paired rod-like pathogenic RNA, is strongly retarded, presumably because of greater flexibility and/or stable curvature. Depending on the gel system, nonlinear extrapolation to 0% T leads to an apparent contour length of 140-230 bp, whereas 130 +/- 20 bp can be determined from electron micrographs and 123-126 bp from secondary structure modeling. We attribute the variation of the electrophoretic behavior of both dsRNA and viroid RNA to interactions with the gel matrix. Nevertheless, extrapolation of the apparent contour length (in bp dsRNA) determined from low-crosslinked polyacrylamide gels (2.6%C) is comparable to the determination by alternative methods.

The structural and functional similarity between viroid and hepatitis delta RNA

The structural and functional similarity between viroid and hepatitis delta RNA

The 5' end Generated in the in vitro Self-Cleavage Reaction of Avocado Sunblotch Viroid RNAs is Present in Naturally Occurring Linear Viroid Molecules

The 5' termini of linear forms of avocado sunblotch viroid (ASBVd) isolated from infected tissue have been determined by primer extension analyses. According to the different termini identified, four main populations of linear viroid molecules were found. One of them had the same 5' end as the one produced in the in vitro self-cleavage reaction of ASBVd dimeric transcripts mediated by a hammerhead structure, suggesting a possible involvement of this in vitro reaction in the in vivo replication mechanism of ASBVd.

Structure of the Maize Mitochondrial Replicon RNA b and Its Relationship with Other Autonomously Replicating RNA Species

The mitochondria of maize plants with S-type cytoplasm possess a family of single and double-stranded RNA molecules, termed RNA plasmids, that replicate in a DNA-independent manner typical of RNA viruses. We have determined the sequence of the smallest and most abundant member of this family, a single-stranded RNA termed RNA b. The 719 nucleotide sequence of RNA b lacks open reading frames of significant length. Probes complementary to the determined (+) strand sequence identified two larger RNA species of 2900 and >4000 nucleotides in S-type maize mitochondria, the smaller of which corresponds to a previously identified member of the RNA plasmid family designated RNA a. These probes also unexpectedly identified a low abundance form of RNA b in N-type maize mitochondria. (+) strand probes identified corresponding (-) strand forms in S cytoplasm that likely represent components of replication intermediates. Primer extension experiments demonstrated that RNA b is an internally deleted form of the larger RNA plasmid, RNA a, and that it has a discrete and homogeneous 5′ terminus. The solubility of RNA b in 2 M-LiCl as well as structural modelling studies indicated that it has a very high degree of secondary structure. Analysis of specific cleavage products generated by treatment of RNA b -oligonucleotide duplexes with RNase H indicated that the only identifiable forms of RNA b were linear molecules with relatively homogeneous 3′ termini; no circular forms were detected. RNA b was found to share a 13 nucleotide sequence with the circular cadang-cadang viroid; 11 of these nucleotides fall within the central conserved region of viroid RNAs. In addition, RNA b shares certain structural similarities with phage Qβ and "variant" RNAs that serve as in vitro templates for the Qβ replicase. Collectively, our results indicate that RNA b is a "defective-interfering" member of an RNA family representing a new and apparently distinct class of RNA replicons.

Mechanism of viroid pathogenesis: Differential activation of the interferon-induced, double-stranded RNA-activated, Mr 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.

Fatal Yellowing of Oil Palms: Search for Viroids and Double-Stranded RNA

Fatal yellowing is a serious disease of still unknown origin affecting oil palms in several regions of Central and South America. In this study a search for viroids and viroid-like RNAs in oil palms was performed using two-dimensional gel electrophoresis and return gel electrophoresis of nucleic acid extracts. Although RNAs showing viroid-like gel-electrophoretic properties were detected, the presence of the known viroids was excluded by hybridization experiments using probes specific for potato spindle tuber viroid (PSTVd), coconut cadang-cadang viroid (CCCVd), or Coleus blumei viroid 1 (CbVd1). By using double-stranded RNA (dsRNA) specific monoclonal antibodies, which do not react with viroid RNA, we were able to show that oil palm RNAs, migrating like viroids are double-stranded RNA species. Since the same dsRNA pattern was found in extracts from diseased as well as from healthy oil palms, the dsRNAs can neither be part of the causative agent of fatal yellowing, nor are they associated with the disease. Their possible origin is discussed. In addition to the standard electrophoretic methods, which have been used for identification of viroids and viroid-like RNAs, we describe additional control experiments to differentiate unequivocally between circular single stranded and linear dsRNA.

Processing of linear longer-than-unit-length potato spindle tuber viroid RNAs into infectious monomeric circular molecules by a G-specific endoribonuclease

Different cDNA constructs were used for the in vitro synthesis of RNA transcripts that contain a complete monomeric unit of the potato spindle tuber viroid (PSTVd) plus an additional repeat of a part of the circular RNA genome. These permutated linear longer-than-unit-length PSTVd RNAs were incubated with the G-specific endoribonuclease RNase T1 which generated monomeric circular PSTVd RNA molecules that were infectious when mechanically inoculated to tomato plants. Besides the correct monomeric PSTVd RNA, smaller and larger circular RNAs were also formed during the reaction. The comparison of different transcripts revealed that correct in vitro processing of PSTVd RNA can proceed at alternative sites indicating that circularization is driven by RNA structure and not governed by a particular sequence. Based on these data, we propose a novel model for the processing of multimeric replicative viroid RNA intermediates through RNA cleavage and ligation catalyzed by a host endoribonuclease.

Highly sensitive digoxigenin-labelled DNA probe for the detection of potato spindle tuber viroid

A molecular probe pSPAv6.2(+), with concatameric insert representing 6.2-times repeated copy of potato spindle tuber viroid (PSTV) RNA, was labelled with digoxigenin and used to detect PSTV by dot-blot hybridization assay. The probe was highly sensitive and specific, detecting as little as 2.5 pg of PSTV RNA. Both severe and mild PSTV strains were detectable in 64–512-times diluted crude extracts from infected tomato leaves, and potato leaves, sprouts, and seeds. For extraction of plant tissue three buffers were compared to determine the lowest non-specific background and the highest sensitivity. The results showed that the digoxigenin-labelled probe is as sensitive as the 32P-labelled probe and can replace radioactive techniques in PSTV detection. With such high sensitivity, the probe is also potentially useful for detecting the viroid in composite samples of mass-indexing programs.

Characterization of RNAs specific to avocado sunblotch viroid synthesized in Vitro by a cell-free system from infected avocado leaves

Analysis by molecular hybridization of the RNAs transcribed by a cell-free fraction from avocado infected with avocado sunblotch viroid (ASBV) demonstrated the presence of newly synthesized viroid-specific sequences, most of which were of the same polarity as the mature infectious viroid RNA. Treatment of the cell-free fraction with DNase reduced the total synthesis of RNA considerably, but it did not influence that of the ASBV-specific RNAs, indicating that the latter were transcribed on an RNA template. Inhibition studies with α-amanitin showed that the synthesis of ASBV-specific RNAs was not affected by concentrations of 1 and 200,μg/ml of the drug, which typically inhibit RNA polymerase I I and III, respectively, from most animal and plant systems. These results suggest that either RNA polymerase I or an unidentified RNA polymerase activity resistant to α-amanitin, acting on an RNA template, plays a role in the replication of ASBV, whereas for the rest of the viroids studied so far it appears that RNA polymerase II is involved. Analysis by polyacrylamide gel electrophoresis under partially and fully denaturing conditions of the ASBV-specific RNAs synthesized in vitro showed that they contain unit and longer than unit length viroid strands, probably associated in complexes with single- and double-stranded regions. The structural properties of these complexes are similar to those of the RNAs accumulating in vivo in viroid-infected tissues, which are the postulated replicative intermediates of the rolling-circle mechanism proposed for viroid synthesis.

Generation of viroid conformational isomers that are stable to incubation with magnesium ions and in a nuclear extract from tomato plants.

We identified conditions for heating and quick cooling viroid RNAs in the presence of salt which lead to the production of conformational isomers stable to incubation for at least 45 minutes at 30 degrees in the presence of magnesium ions. Elution in 0.3 M NaCl allowed the purification of an electrophoretically slow form of an in vitro transcript carrying a complete copy of the potato spindle tuber viroid RNA sequence. Slow forms of this transcript and of kinase-labeled linear viroid RNA persisted for longer than 20 minutes when incubated with a protein-rich extract prepared from the nuclei of uninfected tomato plants, although both were slowly cleaved by a nuclease present in this extract. The fast form of the transcript was highly resistant to this tomato ribonuclease. The slow form of the transcript was much more susceptible to cleavage by RNase T1 than the fast form of this RNA, suggesting that the reduced gel mobility of the slow forms results from their relatively open structure. The ability to purify viroid conformational isomers from polyacrylamide gels will facilitate biochemical studies aimed at identifying host components interacting with RNAs of the viroid replication complex, which may not all be present in the most thermodynamically favored rodlike structure of mature viroids.

The strands of both polarities of a small circular RNA from carnation self-cleave in vitro through alternative double- and single-hammerhead structures.

The sequence of a circular RNA from carnation has been determined and found to consist of 275 nucleotide residues adopting a branched secondary structure of minimum free energy. Both plus and minus strands of this RNA can form the hammerhead structures proposed to mediate the in vitro self-cleavage of a number of small infectious plant RNAs and the transcript of satellite 2 DNA from the newt. Minus full- and partial-length transcripts of the carnation circular RNA including the hammerhead structure showed self-cleavage during transcription and after purification, indicating the involvement of a single-hammerhead structure in the self-cleavage reaction. In the case of the plus transcripts only a dimeric RNA, but not a monomeric one, self-cleaved efficiently during transcription and after purification, strongly supporting the implication in this process of a double-hammerhead structure theoretically more stable than the corresponding single cleavage domain. However, a plus monomeric transcript self-cleaved after purification at a slow rate in a concentration-independent reaction which most probably occurs through an intramolecular mechanism. Comparative sequence analysis has revealed that the circular RNA from carnation shares similarities with some representative members of the viroid and viroid-like satellites RNAs from plants, suggesting that it is a new member of either these two groups of small pathogenic RNAs.

Involvement of nuclear DNA-dependent RNA polymerases in potato spindle tuber viroid replication: a reevaluation

Inhibitor studies on Potato Spindle Tuber Viroid (PSTVd) synthesis were performed with highly purified nuclei from suspension cultures of PSTVd-infected Solanum demissum cells in order to investigate which nuclear RNA polymerases are involved in PSTVd replication. The in vitro synthesized RNA was analyzed by dot blot hybridization with cDNA probes specific for the viroid RNA and for three cellular RNA species transcribed by the nuclear DNA-dependent RNA polymerases I, II and III, respectively. Application of different concentrations of α-amanitin revealed that PSTVd(+) and (−)RNA synthesis was inhibited at exactly the same toxin concentrations as the synthesis of the wun-2 mRNA, which is trancribed by DNA-dependent RNA polymerase II. RNA transcribed by DNA-dependent RNA polymerase I or III was not inhibited at that concentration of α-aminitin. On the hand, PSTVd synthesis was not inhibited by actinomycin D even at high concentrations, indicating that PSTVd replication does not depend on the active transcription of chromosomal DNA. Our results demonstrate that the DNA dependent RNA polymerase II is directly involved in PSTVd(+) and (−)RNA synthesis.

Replication and evolution of viroid-like pathogens.

It is becoming increasingly evident that certain basic processes of macromolecular biology require RNA. Whether as a key component of ribosomes in the translation process, or as small nuclear RNAs in particles carrying out eukaryotic messenger RNA (mRNA) splicing, RNA—perhaps because of its historical role in molecular evolution—occupies a central position. Another class of RNA molecules—the pathogenic viroid-like RNAs—are also providing new insights into the role of RNA in gene expression. Until recently, these circular, self-replicating RNAs—250–400 bases in length—were found only in plants. Beginning in 1986, however, several reports have confirmed that the causative agent for delta hepatitis in mammals contains a circular, viroid-like RNA (Wang et al. 1986; Kos et al.1986; Chen et al. 1986; Taylor et al. 1987). This delta RNA is about four times the size of plant viroid RNAs. Knowledge that such RNAs can proliferate within cells has come at the same time as an increasing interest in the role of RNA in the evolution of primitive self-replicating systems. Several proposals based on RNA—a material with both template and enzymatic properties—as the initial genetic substance have appeared (Gilbert 1986; Sharp 1985).