Nucleotide sequence and secondary structure of apple scar skin viroid.

The complete nucleotide sequence of apple scar skin viroid(ASSV) has been established, and a probable secondary structure is proposed. A single-stranded circular ASSV RNA consists of 330 nucleotides and can assume the rodlike conformation with extensive base-pairing characteristic of all the known viroids. ASSV shows low sequence homologies with other viroids and lacks the central conserved region. These indicate that ASSV should be allocated to a separate viroid group. However, homologous sequences with potato spindle tuber viroid(PSTV) in ASSV occur in limited and scattered regions of both viroids. These homologous regions fall within the particular domains in the viroid domain model which has been previously proposed by Keese and Symons(Proc. Natl. Acad. Sci. USA. 82, 4582-4586, 1985).     

Nucleotide sequence and proposed secondary structure of Columnea latent viroid: a natural mosaic of viroid sequences.

The Columnea latent viroid (CLV) occurs latently in certain Columnea erythrophae plants grown commercially. In potato and tomato, CLV causes potato spindle tuber viroid (PSTV)-like symptoms. Its nucleotide sequence and proposed secondary structure reveal that CLV consists of a single-stranded circular RNA of 370 nucleotides which can assume a rod-like structure with extensive base-pairing characteristic of all known viroids. The electrophoretic mobility of circular CLV under nondenaturing conditions suggests a potential tertiary structure. CLV contains extensive sequence homologies to the PSTV group of viroids but contains a central conserved region identical to that of hop stunt viroid (HSV). CLV also shares some biological properties with each of the two types of viroids. Most probably, CLV is the result of intracellular RNA recombination between an HSV-type and one or more PSTV-type viroids replicating in the same plant.     

Australian grapevine viroid--evidence for extensive recombination between viroids.

Australian grapevine viroid (AGV, 369 residues) is a novel viroid with less than 50% sequence similarity with any known viroid. Nevertheless its entire sequence can be divided into regions, each with a high sequence similarity with segments from one of citrus exocortis, potato spindle tuber, apple scar skin, and grapevine yellow speckle viroids. AGV contains the entire central conserved region of the apple scar skin viroid group and is proposed as a member of this group. AGV appears to have originated from extensive RNA recombination involving other viroids. The vegetatively propagated grapevines which have been exposed to multiple viroid infections during their long history of cultivation may have allowed such recombination.     

Chrysanthemum stunt viroid: primary sequence and secondary structure.

The sequence of the 356 nucleotide residues of chrysanthemum stunt viroid (CSV) has been determined. Overlapping linear viroid fragments were obtained by partial ribonuclease digestion, radiolabelled in vitro at their 5'-ends, and sequenced using partial enzymic cleavage methods. Of the CSV sequence, 69% is contained in the published sequence of potato spindle tuber viroid (PSTV). Differences in the primary sequence of CSV and PSTV suggest that neither the positive nor putative negative strands of these two viroids code for functional polypeptide products. However, the two viroids can form similar secondary structures, implicating a role for viroid structure in replication.     

Hop stunt viroid: molecular cloning and nucleotide sequence of the complete cDNA copy

The complete cDNA of hop stunt viroid (HSV) has been cloned by the method of Okayama and Berg (Mol.Cell.Biol.2,161-170. (1982] and the complete nucleotide sequence has been established. The covalently closed circular single-stranded HSV RNA consists of 297 nucleotides. The secondary structure predicted for HSV contains 67% of its residues base-paired. The native HSV can possess an extended rod-like structure characteristic of viroids previously established. The central region of the native HSV has a similar structure to the conserved region found in all viroids sequenced so far except for avocado sunblotch viroid. The sequence homologous to the 5'-end of U1a RNA is also found in the sequence of HSV but not in the central conserved region.     

Some properties of the viroid inducing peach latent mosaic disease

Analysis by polyacrylamide gel electrophoresis of nucleic acid extracts from different peach samples, healthy or infected with the peach latent mosaic (PLM) disease, demonstrated the association of this disease with an RNA exhibiting the electrophoretic properties typical of circular viroid molecules. This RNA was called peach latent mosaic viroid (PLMV), since a purified preparation of it, when inoculated into GF 305 peach seedlings induced characteristic symptoms of PLM disease. PLMV was estimated to have a molecular size in the range of 330–340 bases, by comparison of its electrophoretic mobility under denaturing conditions with those of several viroid RNA. Dot-blot analysis showed that PLMV has a sequence clearly different from other viroids, including citrus exocortis viroid, apple scar skin viroid (ASSV), hop stunt viroid (HSV) and avocado sunblotch viroid. The possible significance of the limited sequence homology shared by PLMV with HSV, and especially with ASSV, is discussed.     

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.     

Avocado sunblotch viroid: primary sequence and proposed secondary structure.

The sequence of the 247 nucleotide residues of the single strand circular RNA of avocado sunblotch viroid (ASBV) was determined using partial enzymic cleavage methods on overlapping viroid fragments obtained by partial ribonuclease digestion followed by 32p-labelling in vitro at their 5'-ends. ASBV is much smaller than potato spindle tuber viroid (PSTV; 359 residues) and chrysanthemum stunt viroid (CSV; 356 residues). A secondary structure model for ASBV is proposed and contains 67% of its residues base paired. In contrast to the extensive (69%) sequence homology of CSV with PSTV, only 18% of the ASBV sequence is homologous to PSTV and CSV. There are eight potential polypeptide translation products with chain lengths from 4 to 63 amino acid residues coded for by the plus (infectious) strand and four potential translation products (2 to 60 residues) coded for by the minus strand. An improved method is described for the synthesis of gamma-32p-ATP of high specific activity.     

Nucleotide sequence of cucumber pale fruit viroid: homology to hop stunt viroid.

Double stranded cDNA of cucumber pale fruit viroid ( CPFV ) has been cloned by the method of Okayama and Berg (Mol.Cell.Biol.2,161-170 (1982] and the complete nucleotide sequence was established. The covalently closed circular molecules of single-stranded CPFV RNA consists of 303 nucleotides. The nucleotide sequence of CPFV was compared with the previously established sequence of hop stunt viroid (HSV), which consists of 297 nucleotides ( Ohno et al. Nucleic Acid Res.11,6185-6197 (1983]. CPFV differs from HSV in the nucleotide sequence at 16 positions which include 8 exchanges, 7 insertions and 1 deletion. Both viroids share about 95% sequence homology. Considering the pathogenic properties of both viroids together, it is concluded that CPFV is a cucumber isolate of HSV.     

Site-specific mutagenesis of potato spindle tuber viroid cDNA:

Summary The infectivity of cloned viroid cDNAs permits investigation of structure/function relationships in these unusual pathogenic RNAs by systematic site-specific mutagenesis of the cDNAs and subsequent bioassay. We have used three different strategies to create nucleotide substitutions within premelting region 2, a region of potato spindle tuber viroid (PSTV) believed to be important in viroid replication: sodium bisulfitecatalyzed deamination of deoxycytosine residues, oligonucleotide-directed mutagenesis, and construction of chimeric viroid cDNAs from fragments of infectious PSTV and tomato apical stunt viroid cDNAs. Although their effects upon the rod-like native structure of PSTV should be minimal, C U transitions at positions 92 or 284 appeared to be lethal. When inoculation with PSTV cDNA containing a single nucleotide substitution was mediated by the Ti plasmid of Agrobacterium tumefaciens, PSTV progeny with an unaltered wild type sequence was obtained. Two factors, the high error frequency characteristic of RNA synthesis and the use of a systemic bioassay for PSTV replication, may explain such sequence reversion and emphasize the importance of an appropriate bioassay system for screening mutant viroid cDNAs.     

The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops.

A new viroid which does not seem to produce any symptoms of disease, and is therefore tentatively named hop latent viroid (HLV) was found to occur worldwide in hops. HLV proved to be infectious when mechanically inoculated onto viroid- and virus-free hops. The viroid nature of HLV was also substantiated by sequence analysis which revealed that HLV is a circular RNA consisting of 256 nucleotides, that can be arranged into the viroid-specific, rod-like secondary structure. HLV also contains the central conserved region typical for most of the presently known viroids. However HLV does not contain the viroid-specific oligo(A) stretch in the upper left part of its rod-like molecule. Because of this feature and a sequence similarity with the prototypes of the other viroid groups below 55%, HLV can be regarded as the first member of a new viroid group.     

Correlation between structure and pathogenicity of potato spindle tuber viroid (PSTV)

Sequence analysis by primer-extension at the level of their cDNA showed that the RNA genomes of various field isolates of potato spindle tuber viroid (PSTV) of different virulence differ from each other only in a few nucleotides in two distinct regions of the rod-shaped molecule. Despite insertions and deletions the chain length of 359 nucleotides is strictly conserved in all the isolates studied. Thermodynamic calculations revealed that due to the observed sequence differences the region located at the left hand part of the rod-like secondary structure of the PSTV molecule, denoted 'virulence modulating (VM) region', becomes increasingly unstable with the increasing virulence of the corresponding isolate. Based on these data we propose in molecular terms a model for the mechanism of viroid pathogenicity. It implies that the nucleotides of the VM region specify and modulate the binding- and hence the competition-potential of the PSTV RNA molecule for a still unknown host factor(s) and thus determine the virulence of PSTV.     

Determination of potato spindle tumor viroid and chrysanthenum stund viroid using biotinylated olideoxyribonucleotides]

A 26 base long oligodeoxyribonucleotide complementary to a common RNA sequence of potato spindle tuber viroid (PSTV) and chrysantemum stunt viroid (CSV) was synthesized. The 3'-end biotinylated one was used for the detection of PSTV and CSV RNA immobilized on nitrocellulose filters by nucleic acid hybridization. Visualization of hybridization results was performed by two ways, either by streptavidin-alkaline phosphatase conjugate or streptavidine and biotinylated alkaline phosphatase. It was possible to detect 0.65 ng of purified CSV and PSTV RNA. The suggested system of viroid diseases detection can be used by agricultural and horticultural enterprises.