Viroid RNA Research Articles

The viroid and viroid-like RNA database

This is an online database to facilitate research on viroid, viroid-like RNAs and human hepatitis delta virus (vHDV) by presenting a large number of sequences and related data in a comprehensive and user-friendly format (e.g. position of their self-catalytic domains, open reading frame of the vHDV, prediction of the most stable secondary structures, etc.). Most of these RNA species share a common proposed replication pattern known as a DNA-independent rolling circle mechanism. Together, these species form the 'brotherhood' of the smallest known auto-replicable RNAs. This online database is available on the World Wide Web at http://www.callisto.si.usherb.ca/jpperra

Synthesis of Infectious Viroids and Other Circular RNAs

Viroids are small autonomously replicating RNAs that share structural features with other subviral circular single-stranded RNAs of plants. Viroids and other circular single-stranded RNAs can be synthesised in vitro by a PCR-based procedure using a simple set of reactions. Two end-to-end primers are selected from a desired region of the viroid, one for the synthesis of the first strand cDNA and another for the production of the second strand DNA. The second primer contains an 18 nucleotide T7 promoter at its 5' end, and is selected such that the G nucleotide at the transcription start site represents a G in the viroid. Linked reverse transcription-PCR results in linear double-stranded DNA consisting of the viroid sequence and the T7 promoter. Run-off transcription of the PCR product allows the synthesis of exact-length linear viroid RNA which can be circularised by T4 RNA ligase following an enzymic modification of the 5' triphosphate to a monophosphate. This procedure results in authentic viroid molecules and obviates the need for construction and cloning of DNA in the form of tandem repeats for infectivity tests. It also allows PCR-based manipulation of circular RNAs, thus greatly simplifying structure-function analyses of viroid molecules.

The dimerization domain of potato spindle tuber viroid, a possible hallmark for infectious RNA.

Covalently closed circular (+) RNA of the potato spindle tuber viroid (PSTVd) can efficiently dimerize noncovalently upon heating and slow cooling in the presence of monovalent cations or Mg2+. In vitro transcription of subgenomic fragments reveals that the ability to dimerize resides in the "upper strand" of its self-complementary rod-like structure. Nuclease probing of these fragments, namely, molecules spanning either the upper or the lower strand of PSTVd, confirms the existence of the previously proposed hairpins I-III, of which hairpin I might contain noncanonical G.A and A.A base pairs. In addition, the upper and lower (+) strands contain large hairpin loops consisting of stretches rich in either adenosine or uridine. Dimerization of the upper (+) strand results in a nuclease-resistant core encompassing hairpin I and is inhibited by an antisense oligonucleotide spanning the entire hairpin; this palindromic domain thus represents the dimerization site. When upper and lower strands were heated and cooled together, no annealing to a viroid-like duplex of both molecules occurs, only dimerization of the upper strand. Therefore, the dimerization hairpin of viroid RNA represents a unique conformational signal that is homologous to similar regions in the human immunodeficiency virus and other retroviruses.

Precisely full length, circularizable, complementary RNA: an infectious form of potato spindle tuber viroid.

The replication of many viral and subviral pathogens as well as the amplification of certain cellular genes proceeds via a rolling circle mechanism. For potato spindle tuber (PSTVd) and related viroids, the possible role of a circular (-)strand RNA as a template for synthesis of (+)strand progeny is unclear. Infected plants appear to contain only multimeric linear (-)strand RNAs, and attempts to initiate infection with multimeric (-)PSTVd RNAs generally have failed. To examine critically the infectivity of monomeric (-)strand viroid RNAs, we have developed a ribozyme-based expression system for the production of precisely full length (-)strand RNAs whose termini are capable of undergoing facile circularization in vitro. Mechanical inoculation of tomato seedlings with electrophoretically purified (-)PSTVd RNA led to a small fraction of plants becoming infected whereas parallel assays with an analogous tomato planta macho viroid (-)RNA resulted in a much larger fraction of infected plants. Ribozyme-mediated production of (-)PSTVd RNA in transgenic plants led to the appearance of monomeric circular (-)PSTVd RNA and large amounts of (+)PSTVd progeny. No monomeric circular (-)PSTVd RNA could be detected in naturally infected plants by using either ribonuclease protection or electrophoresis under partially denaturing conditions. Although not a component of the normal replicative pathway, precisely full length (-)PSTVd RNA appears to contain all of the structural and regulatory elements necessary for initiation of viroid replication.

Recognition of DNA, RNA, and Proteins by Circular Oligonucleotides

While the backbone organization of DNA and RNA is normally quite regular, there are many structural forms which these molecules can adapt by undergoing various secondary and tertiary helical and folded interactions, by being subjected to twisting and bending, and by interconversion between various topological and knotted variations. Probably the most common structural form of DNA in nature is a long double helix in which the ends are joined into a circle. The genomes of most lower organisms are organized in this way,1 and clearly this circular structure lends some important advantage to these organisms or evolution would not have selected for it. Most chemists are chiefly interested in structure and reactivity of molecules smaller than entire bacterial genomes; such large circular DNAs will not be discussed herein for that reason. Of particular interest, then, is the question: what are the smallest cyclic DNAs or RNAs which exist in nature? For DNA, the answer appears to be several hundred to perhaps 1500 nucleotides (nt), which is still rather large. For RNA, probably the smallest known circular structures are the viroid RNAs, which are single-stranded and as small as 246 nucleotides in size.2 However, this is far from the smallest possible cyclic nucleic acid structure. Duplex DNA can exist in circles at least as small as 125 bp (base pairs);3 cyclic structures smaller than this are difficult to achieve because of the rigidity of the double helix.4 Single-stranded DNA and RNAs do not have this problem, and rings as small as two nucleotides are known.5 Thus, the realm of possible cyclic DNA and RNA structures falls easily into the size range most palatable to chemists. Interestingly, although small synthetic circular DNAs had been reported a number of times as early as in 1968,6 prior to 1990 there were no reported studies investigating the effect of this quite significant structural modification on DNA’s molecular recognition properties. Despite this, there was quite reasonable precedent that such a structural alteration might have a large effect on such recognition properties. Indeed, in recent decades it has become widely recognized that macrocyclic molecular structures can have strong advantages in the formation of noncovalent complexes.7 Among these advantages (relative to noncyclic molecules of similar structure) are tighter binding affinity and greater specificity for binding the target of interest. The advent of simple synthetic approaches to the construction of oligonucleotides has led to an explosion of studies aimed at studying and modifying their noncovalent binding properties. In our early work we proposed that the principle of macrocyclic recognition could also be applied to nucleic acids in small synthetic well-defined systems. The testing of that hypothesis is the subject of this Account.

In vitro and in vivo self-cleavage of a viroid RNA with a mutation in the hammerhead catalytic pocket.

Peach latent mosaic viroid (PLMVd) can adopt hammerhead structures in both polarity strands. In the course of a study on the variability of this viroid a natural sequence variant has been characterized in which the hammerhead structure of the plus polarity strand has the sequence CCGA instead of the conserved uridine turn motif CUGA present in the catalytic pocket of all natural hammerhead structures. The viroid RNA containing this mutant hammerhead structure, but not those with the two other possible substitutions, U-->A and U-->G, in the same position of the catalytic pocket, showed significant self-cleavage activity during in vitro transcription. Moreover, the corresponding full-length PLMVd cDNA was infectious and the mutation was retained in a fraction of the viroid progeny. These results indicate that the sequence flexibility of the hammerhead structure, acting in vitro and in vivo , is higher than anticipated and provide relevant data for a deeper insight into the catalytic mechanism of this class of ribozymes and into the structure of the uridine turn motif.

Inosine 5'-triphosphate can dramatically increase the yield of NASBA products targeting GC-rich and intramolecular base-paired viroid RNA.

Nucleic acid sequence-based amplification (NASBA) according to the standard protocol failed to amplify cRNA of viroids, probably because of their GC-rich and intramolecular base-paired structure. However, NASBA in the presence of inosine 5'-triphosphate successfully amplified the cRNAs to viroids in total nucleic acid extracts from citrus plants. As sequence specificity of the cRNA to viroids was confirmed by northern analysis, the amplification and fidelity of cRNAs are sufficient for the sensitive and specific detection of viroids.

Dynamic competition between alternative structures in viroid RNAs simulated by an RNA folding algorithm

The folding pathways of viroid RNAs were studied using computer simulations by the genetic algorithm for RNA folding. The folding simulations were performed for PSTVd RNAs of both polarities, using the wild-type sequence and some previously known mutants with suggested changes in the stable or metastable structures. It is shown that metastable multihairpin foldings in the minus strand replicative intermediates are established due to the specific folding pathway that ensures the absence of the most stable rod-like structure. Simulations of the PSTVd minus strand folding during transcription reveal a metastable hairpin, formed in the left terminal domain region of the PSTVd. Despite high sequence variability, this hairpin is conserved in all known large viroids of both subgroups of PSTVd type, and is presumably necessary to guide the folding of the HPII hairpin which is functional in the minus strand. The folding simulations are able to demonstrate the changes in the balance between metastable and stable structures in mutant PSTVd RNAs. The stable rod-like structure of the circular viroid (+) RNA is also folded via a dynamic folding pathway. Furthermore, the simulations show that intermediate steps in the forced evolution of a shortened PSTVd replicon may be reconstructed by a mechanistic model of different folding pathway requirements in plus- and minus-strand RNAs. Thus the formation of viroid RNA structure strongly depends on dynamics of competition between alternative RNA structures. This also suggests that the replication efficiency of viroid sequences may be estimated by a simulation of the folding process.

The viroid and viroid-like RNA database.

The viroid and viroid-like RNA database is a compilation of all natural sequences published in journals or available from the GenBank and EMBL nucleotide sequence libraries. Several information regarding these RNA species such as the position of their self-catalytic domains and the open reading frame of the human hepatitits delta virus are provided. The database also includes a determination of the likely ancestral sequence of most species and a prediction of the most stable secondary structures of these sequences. This online database is available on the World Wide Web (http://www.callisto.si.usherb.ca/[symbol: see text]jpperra ). It should provide an excellent reference point for further phylogenetic and structure-function studies of these RNA species.

Synthesis and two-dimensional electrophoretic analysis of mixed populations of circular and linear RNAs

Spontaneous cleavage of the less abundant form of tobacco ringspot virus satellite RNA is readily reversible. Capitalizing on earlier observations by Feldstein and Bruening that small 'mini-monomer' RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are able to efficiently circularize, we have constructed a series of self-circularizing RNAs of precisely known size. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nt could be completely resolved using a novel two-dimensional denaturing polyacrylamide gel electrophoresis system. Similar analyses of a complex mixture of coconut cadang-cadang viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infected palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.

Chrysanthemum chlorotic mottle viroid: unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes.

The causal agent of chrysanthemum chlorotic mottle (CChM) disease has been identified, cloned, and sequenced. It is a viroid RNA (CChMVd) of 398-399 nucleotides. In vitro transcripts with the complete CChMVd sequence were infectious and induced the typical symptoms of the CChM disease. CChMVd can form hammerhead structures in both polarity strands. Plus and minus monomeric CChMVd RNAs self-cleaved during in vitro transcription and after purification as predicted by these structures, which are stable and most probably act as single hammerhead structures as in peach latent mosaic viroid (PLMVd), but not in avocado sunblotch viroid (ASBVd). Moreover, the plus CChMVd hammerhead structure also appears to be active in vivo, because the 5' terminus of the linear plus CChMVd RNA isolated from infected tissue is that predicted by the corresponding hammerhead ribozyme. Both hammerhead structures of CChMVd display some peculiarities: the plus self-cleaving domain has an unpaired A after the conserved A9 residue, and the minus one has an unusually long helix II. The most stable secondary structure predicted for CChMVd is a branched conformation that does not fulfill the rod-like or quasi-rod-like model proposed for the in vitro structure of most viroids with the exception of PLMVd, whose proposed secondary structure of lowest free energy also is branched. The unusual conformation of CChMVd and PLMVd is supported by their insolubility in 2 M LiCl, in contrast to ASBVd and a series of representative non-self-cleaving viroids that are soluble under the same high salt conditions. These results support the classification of self-cleaving viroids into two subgroups, one formed by ASBVd and the other one by PLMVd and CChMVd.

New Insights into the Mechanisms of RNA Recombination

RNA plays many different roles in biological systems. characteristic marker mutations from the parental RNAs. One of RNAs most intriguing features is the ability to Following the initial recombination event, detection of carry genetic information despite a labile nature. While recombinants required amplification in cells that imRNA genomes were presumably widespread in the priposed a selection for or against a particular recombinant mordial ‘‘RNA world,’’ only RNA viruses and viroids conbased on its ability to be amplified in vivo. Nevertheless, tinue to exploit RNA-based genetic materials. As with these data were used to group RNA recombination as DNA-based organisms, these entities must evolve and homologous or nonhomologous (King, 1988). Homoloadapt in order to survive. Genetic recombination, the forgous recombinants were those derived from parental mation of chimeric molecules from segments previously RNAs that were either very similar in the case of intraseparated on the same molecule or present on different typic recombination or somewhat less similar in the case parental molecules, is one of the most important mechaof intertypic recombination. Based on the precision of nisms for generating novel genomes that may have sethe event, Lai (1992) further divided this grouping of relective advantages over parental genomes. Recent studcombinants into homologous or aberrant homologous ies on the evolution of RNA viruses have revealed that recombinants. Homologous recombinants were newly RNA recombination is a widespread phenomenon that defined as containing no sequence alterations (except has shaped modern viruses by rearranging viral gethe existing marker mutations) when compared to the nomes or disseminating functional modules among difparental molecules while aberrant homologous recombiferent viruses (Strauss and Strauss, 1988; Dolja and Carnants contained modifications such as mismatch mutarington, 1992; Lai, 1992; Simon and Bujarski, 1994). In tions, deletions, or insertions at or close to the junction addition, an important short-term function of genetic resite. Recombinants were grouped as nonhomologous if combination may be the rescue of functional sequences they were generated by recombination between dissimifrom mutated parental molecules, which is of particular lar viral genomes or between a viral genome and host significance given the high mutation rates associated RNA. This classical genetic grouping of recombinants, with replication by RNA-dependent RNA polymerases however, did not take into account the mechanisms lead(RdRp) (Domingo et al., 1996). Depending on the preciing to their generation. Not surprisingly, recent studies sion of the repair mechanism, the repaired genome can have suggested similar recombinants can be formed by be similar to the parental genome, or it can contain furdifferent mechanisms. Moreover, similar mechanisms ther mutations. This illustrates that sequence diversity in have been postulated that lead to the formation of some RNA sequences generated by genetic recombination can recombinants that currently would be grouped as homolinvolve both gross changes and minor mutations. Moreogous or nonhomologous. over, the products of recombination can overlap with unThe most accepted models of RNA recombination are related phenomena, including mutagenesis and nonrethe replicase-driven template switching model, the RNA combination-based genome repair. breakage and ligation model, and the breakage-induced Early studies identified RNA recombination events intemplate switching model. Since all these recombination directly by isolating chimeric products that contained models can lead to the formation of similar recombinant RNAs, it is not possible to promote a particular mecha1 To whom correspondence and reprint requests should be adnism based only on the sequence of the recombination dressed to at Department of Biochemistry and Molecular Biology, Ledend-products. Rather, characterization of recombination erle Graduate Research Center, University of Massachusetts, Box intermediates and components of the recombination ma34505, Amherst, MA 01003-4505. Fax: 413-545-4529; E-mail: simon@ biochem.umass.edu. chinery is required. Below, we will summarize our current

Detection and tissue distribution of potato spindle tuberviroid in infected tomato plants by tissue print hybridization.

Potato spindle tuber viroid (PSTVd) was detected in two cultivars of tomato (Lycopersicon esculentum Mill.) by tissue print hybridization of cross-sections of stem and rhachis, using a 35S-labeled PSTVd RNA probe. PSTVd was detectable in the viroid-sensitive and symptom-developping cv "Rutgers" 2 weeks p.i., and in the viroid-tolerant and practically symptomless cv "Goldkugel" 3 weeks p.i. In both tomato cultivars, PSTVd accumulated in the upper parts of the plants newly grown after inoculation. It was predominantly found in association with the ring formed by the vascular tissue. The final accumulation of PSTVd as well as its spatial distribution were similar in the sensitive and in the tolerant tomato cultivar, as estimated from the tissue print autoradiographs. Thus, tissue print hybridization provides a rapid and sensitive means for viroid diagnosis and for the assessment of tissue-specific localization of the viroid RNA.

Evidence for heterologous encapsidation of potato spindle tuber viroid in particles of potato leafroll virus.

The aphid Myzus persicae (Sulz.) was shown to transmit potato spindle tuber viroid (PSTVd) to potato clone DTO-33 from source plants doubly infected with potato leafroll virus (PLRV) and PSTVd. Transmission was of the persistent type and did not occur when the insects were allowed to feed on singly infected plants. Only low levels of PSTVd were associated with purified PLRV virions, but its resistance to digestion with micrococcal nuclease indicates that the viroid RNA is encapsidated within the PLRV particles. Epidemiological surveys carried out at three locations in China revealed a strong correlation between PSTVd infection and the presence of PLRV, suggesting that PLRV can facilitate PSTVd spread under field conditions.

Higher order structures of coxsackievirus B 5' nontranslated region RNA.

A complete understanding of the function of RNA molecules requires a knowledge of their higher order structure (secondary and tertiary) as well as the characteristics of the sequence (primary structure). The two- and three-dimensional structure of RNA is important for many functions, including regulation of transcription and translation, catalysis, and transport of proteins across membranes. Molecules with the same function have the potential to fold into similar structures although they might differ in primary structure, a fact that helps to illustrate the importance of secondary and tertiary structure in relation to function. Examples of such constancy in secondary structure exist in tRNAs, 5sRNAs, 16sRNAs and viroid RNAs. Secondary and tertiary structure of tRNAphe (Kim et al. 1974) and of a hammerhead ribozyme (Pley et al. 1994) have been shown by their crystal structure. Currently little is known of tertiary interactions, but studies on tRNA indicate these are weaker than secondary structure interactions (Riesner and Romer 1973; Crothers and Cole 1978; Jaeger et al. 1990). It is however very difficult to crystallize and/or get nuclear magnetic resonance spectrum data for large RNA molecules. Therefore, a logical place to start in determining the 3-D structure of RNA is determination of the secondary structure.

Update of the viroid and viroid-like sequence database: addition of a hepatitis delta virus RNA section.

Recently, we developed and made available an online database that includes all the reported (to our knowledge) viroid and viroid-like RNA sequences [Bussire,F., Lafontaine,D. and Perreault,J.-P. (1996)Nucleic Acids Res.24, 1793-1798]. We report here an update of this catalogue which includes the addition of a new section devoted to human hepatitis delta virus (vHDV) sequences. This new section comprises all available vHDV sequences, irrespective of their completeness, which have been either published or were available from nucleic acid libraries. Additional structural characteristics of the vHDV genome, such as the positions of the self-catalytic domains, the antigen open reading frames, etc., are also included. The catalogue is available on the World Wide Web (see text) in a user-friendly form. It should provide an excellent reference point for further molecular studies of these small circular pathogenic RNAs.

A simple and sensitive non‐radioactive microplate hybridization for the detection and quantification of picograms of viroid and viral RNA

We have developed a simple and sensitive nucleic acid hybridization assay protocol that is carried out in multi‐well microplates (microplate hybridization), rather than on nitrocellulose or nylon membrane (membrane hybridization). Denatured nucleic acids were directly immobilized on polystyrene microplate, hybridized with a DIG‐labeled cRNA probe, and detected via hydrolysis of p‐nitrophenylphosphate by alkaline phosphatase conjugated with anti‐DIG antibody. The direct microplate hybridization (direct‐MPH) was able to detect subpicogram amounts of viroid cDNA or RNA transcript, as well as native viroid RNA and viral RNA, a level of sensitivity that is comparable to the conventional membrane hybridization. Moreover, the ability to quantitate the results of direct‐MPH using a microplate reader will be convenient for both routine clinical diagnosis and the quantification of small amounts of specific DNAs or RNAs.

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.

A pseudoknot ribozyme structure is active in vivo and required for hepatitis delta virus RNA replication.

The ribozymes of hepatitis delta virus (HDV) have so far been studied primarily in vitro. Several structural models for HDV ribozymes based on truncated HDV RNA fragments, which are different from the hammerhead or the hairpin/paperclip ribozyme model proposed for plant viroid or virusoid RNAs, have been proposed. Whether these structures actually exist in vivo and whether ribozymes actually function in the HDV replication cycle have not been demonstrated. We have now developed an in vivo ribozyme self-cleavage assay capable of detecting self-cleavage of dimer or trimer HDV RNA in vivo. By site-directed mutagenesis and compensatory mutations to disrupt and restore potential base pairing in the ribozyme domain of the full-length HDV RNA according to the various structural models, a close correlation between the detected in vivo and the predicted in vitro ribozyme activities of various mutant RNAs was demonstrated. These results suggest that the proposed in vitro ribozyme structure likely exists and functions during the HDV replication cycle in vivo. Furthermore, the pseudoknot model most likely represents the structure responsible for the ribozyme activity in vivo. All of the mutants that had lost the ribozyme activity could not replicate, indicating that the ribozyme activities are indeed required for HDV RNA replication. However, some of the compensatory mutants which have restored both the cleavage and ligation activities could not replicate, suggesting that the ribozyme domains are also involved in other unidentified functions or in the formation of an alternative structure that is required for HDV RNA replication. This study thus established that the ribozyme has important biological functions in the HDV life cycle.

RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase.

Hepatitis delta virus (HDV) is a subviral human pathogen that requires hepatitis B virus (HBV) for packaging. Concurrent infection by HBV and HDV increases the risk of severe liver disease compared to infection with HBV alone. The HDV genome is a closed circular RNA of about 1,700 bases which is replicated through an RNA intermediate, the antigenome. Both RNAs can be folded into highly base-paired, rod-shaped structures, similar to the plant viroid RNAs. Two forms of the sole HDV protein, hepatitis delta antigen, are derived from a single open reading frame by RNA editing; the enzymes responsible for the editing have not been characterized. Here we report that the purified enzyme dsRAD (for double-stranded-RNA-adenosine deaminase) can edit HDV antigenomic RNA in vitro. Most important, we observe that mutations in critical sequences of the antigenome have identical effects on in vitro and in vivo editing, suggesting that dsRAD, or a closely related enzyme, is responsible for editing HDV RNA in vivo.