Viroids are unencapsidated, small, circular, single-stranded RNAs which replicate autonomously when inoculated into host plants. Some are pathogenic, but others replicate without eliciting symptoms.
Taxonomic Structure of Viroids
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Physical Chemical and Physical Properties
Viroid molecules, with a Mr of 80-125 103, display extensive internal base pairing to give in most cases, in vitro rod-like or quasi-rod-like conformations about 50 nm long (Fig. 1).
These structures denature by cooperative melting (Tm in 10 mM Na+ about 50°C) to single-stranded circles of approximately 100 nm contour length. Viroids can also form metastable structures with hairpins that may be functionally important. However, at least two viroids, Peach latent mosaic viroid (PLMVd) and Chrysanthemum chlorotic mottle viroid (CChMVd), do not follow this rule and they clearly appear to adopt branched conformations. There is experimental support for the contention that PLMVd and CChMVd have unique conformations because they are insoluble in 2 M LiCl as opposed to the rest of viroids which remain soluble under the same high salt conditions. Elements of tertiary structure also exist in viroids. Sequences vary from 246 to 399 nt in length and are rich in G+C (53-60%) with the only exception of Avocado sunblotch viroid (ASBVd) (38%).
Genome Organization and Replication
All viroids, except ASBVd, PLMVd and CChMVd, share a model of five structural-functional domains within the proposed rod-like secondary structure of minimal free energy: C (central), P (pathogenic), V (variable), and TR and TL (terminal right and left). The C domain contains a central conserved region (CCR) formed by two sets of conserved nucleotides located in the upper and lower strands, with those of the upper strand being flanked by an inverted repeat (Fig. 2).
The upper strand of the CCR can form either a hairpin or, in oligomers, a palindromic structure possibly relevant in replication. Two other conserved sequence motifs are the terminal conserved region (TCR), found in all members of the Pospiviroid and Apscaviroid genera and in the two largest members of the Coleviroid genus, and a terminal conserved hairpin (TCH) present in all members of Hostuviroid and Cocadviroid genera (Fig. 2). The conservation of these two motifs in sequence and location in the left terminal domain suggests their involvement in some critical functions. Coconut cadang-cadang viroid (CCCVd) is unusual in occurring as RNAs of different sizes, the larger ones having sequence repetitions of the smallest one; a similar situation has been found recently in Citrus exocortis viroid (CEVd). Some viroids, the most striking examples being Columnea latent viroid (CLVd) and Australian grapevine viroid (AGV), appear to have emerged from intermolecular RNA recombination events since they seem to consist of a more or less complex mosaic of sequences present in other viroids. Direct evidence in support of an ongoing recombination process has come from studies on the dynamic distribution over time of the three Coleus viroids coinfecting individual plants. There is no evidence that viroids encode proteins and unlike viruses which primarily parasitize the host translation machinery, viroids mainly parasitize host transcription, possibly by using RNA polymerase II and/or other cellular RNA polymerases. Multimers isolated from infected tissue may be replicative intermediates produced by a rolling circle mechanism with two variants (asymmetric and symmetric) and three steps (RNA polymerization, cleavage and ligation). ASBVd, PLMVd and CChMVd self-cleave in vitro and very probably in vivo through hammerhead structures to produce unit length strands but others do not, and may rely on host factors for cleavage, although self-cleavage through a different class of ribozymatic structures cannot be excluded (Fig. 3).
No antigenicity demonstrated.
Some viroids have wide host ranges in the angiosperms but others have narrow host ranges. Coconut cadang-cadang viroid (CCCVd) and Coconut tinangaja viroid (CTiVd) infect monocotyledons, the remainder infect dicotyledons. Old cultivars of grapevine and citrus can harbor at least five different viroids
Viroids are transmitted mainly by vegetative propagation. Most are transmitted mechanically and some in seed or pollen. Only Tomato planta macho viroid (TPMVd) is known to be efficiently transmitted by aphids.
Interactions at the level of symptom expression and viroid accumulation have been detected in plants co-infected by two strains of a viroid or even by two different viroids sharing extensive sequence similarities. Interactions of this class have been observed in the case of members belonging to both viroid families (see below), suggesting the possible existence of more than one mechanism of cross protection between viroids.
The presence and type of CCR, and the presence or absence of the two other conserved regions TCR and TCH, are useful criteria for classifying most viroids within the first family Pospiviroidae and for their allocation into the genera. ASBVd, PLMVd and CChMVd form a second family of viroids Avsunviroidae without CCR but endowed with self-cleavage through hammerhead ribozymes (Fig. 3). The type of hammerhead structure, together with the G+C content and the solubility in 2 M LiCl are used to demarcate species into genera. Essentially the same grouping is obtained by using phylogenetic trees derived from the whole sequences (Fig. 4). An arbitrary level of less than 90% sequence similarity and distinct biological properties, particularly host range, currently separates species within genera. Due to the heterogeneous nature of viroid populations it should be expected that a spectrum of closely related variants, having more than 90% sequence similarity, will co-exist within an infected plant, although one or a limited number of them may represent the bulk of the population.
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