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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.

Base Sequence↗

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.

Computer Communication Networks↗

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

Databases, Factual↗

The database of the smallest known auto-replicable RNA species: viroids and viroid-like RNAs.

This is an online database in order to facilitate research on viroid, viroid-like RNAs and human hepatitis delta virus 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, prediction of the most stable secondary structures, etc.). This online database is available on the WWW at http://www.callisto.si. usherb.ca/jpperra

Catalysis↗

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.

Base Sequence↗

Eggplant latent viroid, the candidate type species for a new genus within the family Avsunviroidae (hammerhead viroids).

Viroids, small circular RNAs that replicate independently and in most cases incite diseases in plants, are classified into the families Pospiviroidae, composed of species with a central conserved region (CCR) and without hammerhead ribozymes, and Avsunviroidae, composed of three members lacking CCR but able to self-cleave in both polarity strands through hammerhead ribozymes. Here we report the biological and molecular properties of Eggplant latent viroid (ELVd). Purified circular ELVd induces symptomless infections when inoculated into eggplant seedlings. ELVd can be transmitted horizontally and through seed. Sequencing 10 complete cDNA clones showed that ELVd is a circular RNA of 332 to 335 nucleotides with high variability. This RNA can adopt a quasi-rod-like secondary structure of minimal free energy and alternative foldings that permit formation of stable hammerhead structures in plus and minus strands. The ribozymes are active in vitro and, most likely, in vivo. Considering the ELVd properties to be intermediate between those of the two genera of family Avsunviroidae, we propose ELVd as the type species of a third genus with the name ELAVIROID:

Base Sequence↗

Viroids and viroid-like satellite RNAs: a phylogenetic analysis.

With the discovery that certain RNAs possess catalytic properties (Kruger et al., 1982; Guerrier-Takada et al., 1983), the possible significance of certain small plant pathogenic RNAs, namely viroids and viroid-like satellite RNAs, as relics of precellular evolution deserves consideration. As a first step in this endeavor, I report here the results of a phylogenetic study of the computer-aligned nucleotide sequences of these RNAs that is based on the parsimony principle (Felsenstein and Sober, 1986).

Base Sequence↗

Oligomeric forms of potato spindle tuber viroid (PSTV) and of its complementary RNA are present in nuclei isolated from viroid-infected potato cells.

Different oligomeric forms of PSTV are detected in nuclei isolated from PSTV-infected potato cells by means of molecular hybridization, using as probes synthetic oligodeoxyribonucleotides with sequence specificity for (+)PSTV and for (-)PSTV. In addition to several species of longer-than-unit-length (-)PSTV molecules, two oligomeric forms of (+)PSTV are detected, which correspond in size to RNA strands of approximately two and three times viroid unit-length. They must be considered as the precursors of the circular and linear (+)PSTV monomers accumulating in the cell nucleus.

Base Sequence↗

Nuclear DNA from uninfected or potato spindle tuber viroid-infected tomato plants contains no detectable sequences complementary to cloned double-stranded viroid cDNA.

High molecular weight tomato nuclear DNA was isolated from uninfected and potato spindle tuber viroid (PSTV)-infected tomato leaves. Restriction digests were fractionated on agarose gels, denatured and transferred to diazobenzyloxymethylpaper, and hybridized to 32P-labeled cloned double-stranded PSTV cDNA. No hybridization to DNA from either uninfected or infected tissue could be detected under conditions that permitted detection of cloned double-stranded PSTV cDNA at a concentration equivalent to one-fifth copy of PSTV-related DNA per haploid tomato genome. Hybridization of tomato DNA to 32P-labeled cloned soybean 18S and 28S ribosomal DNA sequences showed that the restricted nuclear DNA was suitable for hybridization to probes containing homologous sequences. Our results indicate that neither PSTV nor its complementary strand is transcribed from nuclear DNA but do not rule out the possibility of sequence homology between host DNA and a small portion of PSTV or its complement.

Base Sequence↗

A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid.

A rapid and sensitive assay was developed for the detection and identification of viroids by standard or multiplex reverse transcription-polymerase chain reaction (RT-PCR)-probe capture hybridization (RT-PCR-ELISA). The assay was applied successfully for the detection and identification of the following six viroid species from infected tissues: Potato spindle tuber viroid (Pospiviroid), Peach latent mosaic viroid (Pelamoviroid), Apple scar skin viroid (Apscaviroid), Apple dimple fruit viroid (Apscaviroid), Pear blister canker viroid (Apscaviroid), and Hop stunt viroid (Hostuviroid). Total RNA was obtained from infected tissue by the Qiagen RNeasy kit and, then viroid cDNA was synthesized using viroid specific complementary DNA primer. To identify and differentiate the amplicons of the six viroids, each amplicon was digoxigenin (DIG)-labelled during the amplification process, and then detected by a colorimetric system using a biotinylated cDNA capture probe specific for each viroid. The results revealed that each capture probe hybridized only to its complementary DIG-labelled amplicon. Thus the six viroids can be detected and differentiated in a multiplex RT-PCR-ELISA assay. In the multiplex assay, cDNAs of six viroids were synthesized simultaneously in one tube, DIG-labelled during amplification, then a portion of the DIG-labelled amplified products was hybridized with selected capture probe. All the six viroid capture probes hybridized to their respective complementary DIG-labelled RT-PCR-amplified product. These findings are important for viroid detection and identification for studying host-viroid interactions and for management and control viroid diseases.

DNA Probes↗

Viroids: molecular infectious agents.

In 1971, unique small RNA molecules, the viroids, were found to cause specific infectious diseases of plants. They are the smallest and simplest contagious agents known. Until now, 14 viroids have been described and 12 diseases of potatoes, tomatoes, citruses, chrysanthemums, cucumbers, hops, coconut palms avocado trees and burdock are known to be caused by viroids. The common symptoms of these diseases are: stunting of plants. discoloration of veins, epinasty, curling and distortions of leaves, chlorotic or necrotic spots etc., followed by death of the diseased plants. All viroids are ssRNAs of m.w. ranging from 1.1 x 10(5) to 1.7 x 10(5), corresponding to chains of just 246 to 371 ribonucleotides. For 10 viroids, complete nucleotide sequences are known PSTV, CSV, CEV, TPMV and TASV show 60%-80% homology with each other; in analogy, ASBV, HSV, CPFV. GV and CCCV are closely homologous to each other, too, but just distantly related to the PSTV group. Extensive intramolecular base pairing creates a characteristic secondary structure of the cyclic viroid RNA chain, native viroids appearing as quasi double-stranded, unbranched, very short rod-like structures with short single-stranded loops. (Thus PSTV forms rods about 50 nm long and 2 nm wide.) The stretch of nearly all viroids bears a common central conserved region of 19 bp. The "upper" part of this region is, presumably, the cleavage-ligation site of viroid oligomers during replication. Viroids are located and replicated in nuclei of infected cells, in association with their nucleoli. Their replication is directed by host DNA-dependent RNA polymerase II using cRNA oligomers as templates according to the rolling circle model. Viroid RNA has no mRNA function. The virulence of viroids is coded by their virulence modulating region in the "left hand" part of their molecules: a single nucleotide substitution between nucleotides 43 and 56 within this region alters the virulence. Most probably, viroids have originated by the circularization of spliced-out transcripts of eucaryotic introns. A stable complex may be created between the 5' end of U1 snRNA and nucleotides 257 to 279 of PSTV cRNA strand; thus the pathogenic effects of viroids seem to be a result of their interference with pre-mRNA processing.

Base Sequence↗