Viroids.pdf

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ViroidsRicardo Flores, Instituto de Biologa Molecular y Celular de Plantas (UPV-CSIC), Valencia, Spain

Jose-Antonio Daros, Instituto de Biologa Molecular y Celular de Plantas (UPV-CSIC), Valencia,Spain

Carmen Hernandez, Instituto de Biologa Molecular y Celular de Plantas (UPV-CSIC), Valencia,Spain

Francesco Di Serio, Instituto de Biologa Molecular y Celular de Plantas (UPV-CSIC), Valencia,Spain

Viroids are infectious small circular RNAs able to induce specific diseases in higher plants.

They differ from viruses in fundamental aspects like structure, function and evolutionary

origin.

Genome Structure

Viroid genomes are exclusively formed by a single-stranded circular ribonucleic acid (RNA), ranging in sizefrom approximately 250 to 400 nucleotides, and being,therefore, the smallest known autonomous replicons(Diener, 1971). Viroid RNAs display a characteristic highdegree of self-complementarity leading to compact con-formations with short single-stranded loops separated bydouble-stranded regions (Gross et al., 1978) that probablyafford some protection against degradation by cellularnucleases. Viroids are able to infect higher plants ofeconomic interest and to incite specific diseases in most ofthem (Table 1). Although the available experimental datastrongly suggest that viroid RNAs, as opposed to virus

genomes, do not code for any protein, they contain all theinformation needed for their survival and propagation.The small size of viroid genomes implies that theinformation embodied in them is extremely compressedand specific regions of the molecule are presumablyinvolved in more than one function. Viroid replication,and most likely viroid movement through the infectedplant, occurs via interactions with host proteins. Thus,these RNA molecules have sequence and structural motifsthat enable them to exploit to their own advantage thecellular machinery whose normal functioning, whenoccasionally impaired, leads to the appearance of thedisease. Additionally, some viroids are catalytic RNAs and

supply ribozymatic activities mediating some steps of theirreplication cycle (see below).

Classification

On the basis of conserved sequence and structural motifsand phylogenetic analysis, viroids have been classified intotwo families, Pospiviroidae and Avsunviroidae, whose typemembers are Potato spindle tuber viroid (PSTVd) andAvocado sunblotch viroid (ASBVd) respectively (Table 1).

Most of the known viroids belongto thefirstfamily and ar

characterized by having some conserved sequence motifsprominent among which is a central conserved regio(CCR) and a rod-like secondary structure. Members of thsecond family, comprising only of ASBVd, Peach latenmosaic viroid (PLMVd) and Chrysanthemum chlorotmottle viroid (CChMVd), do not have a CCR but thstrands of both polarities are able to form hammerheastructures and to self-cleave through these ribozymatiactivities. Within each family viroids are allocated intgenera on the basis of some commonly shared moti(Table 1) and then into species, which have a sequencsimilarity level of less than 90% and specific biologicaproperties, particularly host range.

Secondary structure: structural and functionadomains

Pospiviroidae members adopt a rod-like or quasi-rod-liksecondary structure in vitro, but the in vivo conformatiomay not be necessarily the same because it can binfluenced by interactions with host proteins. Howeverthere is experimental support, at least in some Pospivioidae representatives, for an in vivo quasi-rod-like conformation. Based on comparative pairwise sequencsimilarities between PSTVd and closely related viroids, model was proposed that divides the rod-like structure int

five structural domains (Keese and Symons, 1985(Figure 1). In this model, now extended to all thPospiviroidae family, the central domain (C) is flanked bthe pathogenic (P) and variable (V) domains, and adjacento them are located theleftterminal (TL) and right termina(TR) domains, respectively (Figure1a). Someof the domainhave been associated with specific functions, as illustrateby the case of the P domain that received this name becausnucleotide changes between severe and mild isolates oPSTVd and Citrus exocortis viroid (CEVd) map into th

Article Contents

Secondary article

. Genome Structure

. Interaction with Host

. Replication

. Pathogenesis

. Evolution

. Control

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region. Recent work, however, indicates that the situationis more complex and that other domains are also involvedin triggering symptom expression (Sano et al., 1992).

Within the C domain there is a CCR formed by shortsequences, conserved within each Pospiviroidae genus,located in opposite positions in both strands, with those ofthe upper strand being flanked by imperfect inverted

repeats that contribute to stabilizing some transientstructures that may be important in replication. Althoughthe conserved sequences of both CCR strands are usuallyassumed to interact through canonical WatsonCrick basepairs (Figure 1a), there is experimental evidence indicatingthat this is not thecase, at least in PSTVd,in which an arrayof noncanonical base interactions form a so-called loop E,described previously in the 5S RNA and involved inbinding transcription factor IIIA and ribosomal proteinL5 implicated in 5S RNA transcription and intranuclear

movement, respectively. The possibility exists that PSTVcould also bind these two proteins. The terminal conserveregion (TCR) and the terminal conserved hairpin (TCH(Figure 1a), are two other motifs whose conservation isequence and relative position in the rod-like secondarstructure of different Pospiviroidae genera argue in favouof their important functional role, so far unknown. It i

interesting to note that both motifs have never beeconcurrently found within the same viroid RNA: the TCRis present in viroids with a size greater than approximatel300 nucleotides, whereas viroids with smaller sizes contaithe TCH.

The most peculiar feature of the three viroids forminthe Avsunviroidae family is that they are catalytic RNAthat can self-cleave through hammerhead ribozyme(Figure 1b). From the structural point of view, ASBVdwith an usual low G + C content among viroids and a rod

Table 1 Viroid classification

Family Genus Speciesa Sizeb

Pospiviroidae Pospiviroid Potato spindle tuber viroid (PSTVd) 356, 359360

With CCR Mexican papita viroid(MPVd) 359360

Without hammerhead

self-cleavage

Tomato planta macho viroid(TPMVd) 360

Chrysanthemum stunt viroid(CSVd) 354, 356Citrus exocortis viroid(CEVd) 370375, 463

Tomato apical stunt viroid(TASVd) 360, 363

Iresine viroid 1 (IrVd-1) 370

Columnea latent viroid(CLVd) 370, 372

Hostuviroid Hop stunt viroid (HSVd) 295303

Cocadviroid Coconut cadang-cadang viroid(CCCVd) 246247, 287

301

Coconut tinangaja viroid(CTiVd) 254

Hop latent viroid(HLVd) 256

Citrus viroid IV(CVd-IV) 284

Apscaviroid Apple scar skin viroid (ASSVd) 329330

Citrus viroid III(CVd-III) 294, 297

Apple dimple fruit viroid(ADFVd) 306307Grapevine yellow speckle viroid 1 (GYSVd-1) 366368

Grapevine yellow speckle viroid 2 (GYSVd-2) 363

Citrus bent leaf viroid(CBLVd) 318

Pear blister canker viroid(PBCVd) 315316

Australian grapevine viroid(AGVd) 369

Coleviroid Coleus blumei viroid 1 (CbVd-1) 248, 250251

Coleus blumei viroid 2 (CbVd-2) 301302

Coleus blumei viroid 3 (CbVd-3) 361362, 364

Avsunviroidae Avsunviroid Avocado sunblotch viroid (ASBVd) 246250

With hammerhead

self-cleavage

Pelamoviroid Peach latent mosaic viroid(PLMVd) 335338

Without CCR Chrysanthemum chlorotic mottle viroid(CChMVd) 398399

CCR, central conserved region.a The first viroid in each genus is the type species.b Size in nucleotides of different variants.

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like secondary structure of lowest free energy, clearlydiffers from PLMVd andCChMVd, which assume a highlybranched conformation in vitro (Navarro and Flores,1997). Evidence supporting the biological significance ofthis branched conformation is particularly strong inCChMVd, where some variants present compensatory

mutations preserving the existence of some of the hairpinof the predicted most stable secondary structure. Thesobservations show that the rod-like secondary structure not a universal property of viroids. It must also be notethat the conformation of some viroid RNAs may b

In HSVd andCCCVd genera

In PSTVd and ASSVdgenera, and in CbVd-2

and CbVd-3

TL

TCH TCR

P

UG

C

G

C

G

C

G

CU

AA

CCNNGNGGUUCCUGUGG CU

A

U

A

C

G

A

U

G

CGCU

G

A

G

ACAA

A U

A

C

G

C

GU

C

G

C

G

G

C

G

C

G

C

CCR

C

GAAA

UCAA

CCUGGAG

In PSTVd genus; othersequences in HSVd,CCCVd, ASSVd and

CbVd-1 genera

V TR

C

G

C

G

G

C

G

C

G

CU3

5 GA

AU

AA

CN

A CN

C

N

U

N

G

N

G

N

A

N 5

3

Loop E(a)

GAAAC

AG

GU

AGUC

AC GUUUC

GACU CU

ASBVd

CAAAG AG UGAGUC

ACGUUUC UCAGUC

PLMVd

N NN NN NN NG AA GA NA U

C GN NN NN NN NN N

N N

5 3

3 5

Helix II

Helix III

N

N

N

N

N

N

N

N

N

N

N5

3

A CUG

Helix I

(b)Consensus hammerhead structure

Figure 1 Structural models for viroids. (a) Rod-like secondary structure proposed for members of the Pospiviroidaefamily. The approximate locationsthe fivestructural domains C (central),P (pathogenic),V (variable), andTL andTR (terminalleft andright, respectively)are indicated.Nucleotidesequencof the TCH (terminal conservedhairpin), TCR (terminalconservedregion)and CCR (central conserved region) are shownwithin boxes,togetherwith the

occurrencein differentviroids.Arrows representflankingsequences that,along withthe corenucleotides of the upper CCR strand, formimperfect inverterepeats. Theinset showsloop E withan S-shapedline connectingthe residueslinked afterultravioletirradiation; enlargedlettersrefer to nucleotides thataconservedin differentRNAs containing this structural motif. (b) Rod-like andbranchedconformations proposed for the type members ofAvsunviroidan

Pelamoviroidgenera, respectively. Sequenceconservedin allhammerhead structuresare shownwithinboxes withdark andwhite backgrounds for plusanminus polarities, respectively. A consensus hammerhead structure is presentedwithin the inset, withthe arrowheadmarking the self-cleavage site.In bo

panels N indicates nonconserved residues and continuous lines and dots between them denote canonical and noncanonical base pairs, respectively.ASBVd, Avocado sunblotch viroid; ASSVd, Apple scar skin viroid; CbVd, Coleus blumei viroid; CCCVd, Coconut cadang-cadang viroid; HSVd, Hop stunt viroiPLMVd, Peach latent mosaic viroid; PSTVd, Potato spindle tuber viroid.

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additionally stabilized by tertiary interactions as pseudo-knots, tetraloops and triple helices.

Interaction with Host

The host range of different viroids is very variable. For

example, whereas Hop stunt viroid(HSVd) is able to infecta wide plant spectrum, each member of the Avsunviroidaefamily is restricted to a few closely related species. Themolecular features determining the host range of viroidsare unknown, but they probably depend on specificinteractions of the RNA with host proteins. Recently, ithas been shown that a single nucleotide substitution inPSTVd enables this viroid to infect tobacco. Interestingly,the affected position is located in the lower strand of theCCR and forms part of the loop E (Figure 1a).

Crossprotection

The viroid ability to infect a host plant may relate toprevious infections by other strains of the same, or by aclosely related, viroid. In fact, when a plant is preinfectedwith a mild viroid strain and is then challenge inoculatedwith a severe strain of the same viroid, the typicalsymptoms of the latter strain and the accumulation levelof its corresponding RNA are attenuated for an indeter-minate period, probably as a consequence of the competi-tion between the two RNAs for a limiting host factor;therefore, crossprotection phenomena similar to thosepreviously reported in viruses, also occur in viroids.However, because of the basic structural and functionaldifferences between viruses and viroids, and even between

the two viroid families, a panoply of very distinct under-lying mechanisms can be anticipated.

Viroid movement

For a successfulinfection the viroid RNA must be able notonly to replicate but also to move within the plant. Long-distance PSTVd movement in tomato has been studied andthe results indicate that the viroid follows the movement ofphotosynthetic products through the phloem, the sameroute that is also usedby most plant viruses. Short-distancemovement has been explored by microinjecting PSTVdinto tobacco and tomato mesophyll; the viroid seems to

move cell-to-cell via plasmodesmata and this movementrequires some viroid-specific sequence or structural motifs.In both types of movement the viroid RNA most likelyinteracts with host proteins but there is no direct evidenceof this.

Subcellular localization

Diverse experimental approaches, which include in situhybridization combined with confocal laser scanning and

transmission electron microscopy, have revealed thaseveral members of the Pospiviroidae family accumulatin the nucleus, with PSTVd and Coconut cadang-cadanviroid (CCCVd) predominantly being concentrated in thnucleoli, whereas CEVd is more uniformly distributethroughout the whole nuclear structure (Figure 2). Sincrecent progress indicates that a series of small nuclear an

nucleolar RNAs, that play important roles in cellulametabolism, have localization signals targeting them tthese subcellular compartments, a similar situation probably occurs in members of the Pospiviroidae familyalthough the specific nature of these signals remains to bdetermined. Thesituationis again very different in ASBVdwhich accumulates in the chloroplast and particularly ithe thylakoid membrane. In the case of PLMVd anCChMVd, the two other Avsunviroidae components, thsubcellular localization has not been established due to thtechnical difficulties derived from their very low concentration in infected tissues, but presumably they alsaccumulate in the chloroplast.

Replication

Viroids replicate through RNA intermediates (Grill anSemancik, 1978) and are assumed to follow a rolling circmodel (Branch and Robertson, 1984). This model waproposed in view of the circular nature of the viroimolecule and the presence in infected tissues of lineaoligomeric viroid RNAs of both polarities, the putativreplication intermediates. The model envisages tw

Figure 2 In situ hybridization to Citrus exocortis viroid(CEVd) in tomatoConfocal micrograph of single mesophyll cell from CEVd-infected

tomato leaf material showing cell nucleus with viroid signal (red/orangeand cell structure by autofluorescence (green). Reproduced by permissiofrom Bonfiglioli et al. (1996) The Plant Journal9: 457465.

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alternative pathways, termed asymmetric and symmetric,with one and two operating rolling circles, respectively.

Rolling circle replication: asymmetric variant

Members of the Pospiviroidae family, such as PSTVd,

CEVd, HSVd, and CCCVd, follow this pathway in whichthe infecting monomeric circular RNA (to which byconvention is assigned the plus polarity) is used as atemplate by an RNA polymerase activity that, after severaltranscription rounds, leads to an oligomeric minus RNA.This concatemer is transcribed to produce an oligomericplus RNA, which is then processed by a site-specificRNAase activity into monomeric strands that are finallycircularized by an RNA ligase activity (Figure 3). PSTVdoligomeric RNAs have been detected in the nucleus,indicating that this viroid, and probably the otherPospiviroidae members, not only accumulates but alsoreplicates in this organelle. Concerning the three enzymatic

activities, the RNA polymerase involved in synthesis ofplus strands is very likely the nuclear RNA polymerase II,acting on an RNA template, because replication of HSVd,PSTVd and CEVd is impeded by the low levels of a-amanitin that typically inhibit this class of enzymes. TheRNA polymerase mediating synthesis of the minus strandsmay also be the same enzyme or another nuclear RNApolymerase. On the other hand, extractsfrom potato nucleiareable to process in vitro a linear oligomeric PSTVd RNAinto monomers and to circularize them, but the nature ofthe RNAase and RNAligase presumably involved remainsto be established.

Rolling circle replication: symmetric variant

In this pathway, the oligomeric minus RNA is processed tounit-length strands and ligated to the circular minus RNAthat then serves as the template for the second half of thecycle. Monomeric circular ASBVd RNAs of both pola-rities have been found in infected tissue, forming multi-stranded complexes with oligomeric RNAs of the oppositepolarity, supporting the idea that replication of this viroid,and probably of the other Avsunviroidae members, occursthrough the symmetric pathway (Figure 3). Moreover, theASBVd multistranded complexes have been found inchloroplasts, indicating that viroid replication occurs in

this organelle, in which it also accumulates. Consistentwith this view, the elongation of ASBVd plus strands is notinhibited by high levels of a-amanitin, suggesting that achloroplastic RNA polymerase, resistant to this antibiotic,is involved. Processing of both polarity oligomeric RNAsof ASBVd, PLMVd and CChMVd occurs autocatalyti-cally in vitro through hammerhead ribozymes (Figure 1b).Two lines of evidence suggest this is also the situation invivo. Firstly, the monomeric plus linear CChMVd RNAextracted from infected chrysanthemum has 5- and 3-

termini which are identical to those generated in vitro bythhammerhead structures; a similar situation has beeobserved regarding the 5-termini of some plus and minulinear ASBVd RNAs of sub- and supragenomic sizeSecondly, sequence variability of PLMVd and CChMValways preserves the stability of hammerhead structureeither because mutations are found in loops or, wheoccurring in helices, a second compensatory mutatio

restores the base pairing. Ligation of linear monomeriRNAs to the circular forms occurs autocatalytically ivitro, at least in PLMVd, but the nature of the resultinbonds, 25 insteadof the typical 35, and the structure othe ligation site of one viroid-like satellite RNA that selfcleaves also through a hammerhead structure, makes thinvolvement of a host RNA ligase more likely.

Important issues on viroid replication remain to baddressed, like the precise site of initiation of RNAsynthesis. Moreover, there are conflicting reports abou

+RNA pol II (?)

RNA pol II

RNAase (?)

5 OH

RNA ligase

(a)

+

Chloroplastic RNA pol (?)

Ribozyme

5 OH2P

3

RNA ligase (?)

(b)

Ribozyme

RNA ligase (?)

Chloroplastic RNA pol (?

5 OH3

2

P3

2

P

Figure 3 Rolling circle model for replication of viroids. (a) Asymmetric

and (b) symmetric variants with one and two rolling circles proposed tooperate in the Pospiviroidaeand Avsunviroidaefamilies, respectively. Soliand open lines refer to plus and minus polarities, respectively, andprocessing sites are denoted by triangles. The enzymatic and ribozymati

activities presumablyinvolvedin the replicationsteps are indicated; for th

activities followed by a question mark the evidence is insufficient orcontroversial. The linear monomeric RNAs contain most probably 5 -hydroxyl and 2,3-cyclic phosphate termini.

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the processing site of the oligomeric plus RNAs of closelyrelated Pospiviroidae members, which has been alterna-tively located in the upper and lower CCR strands, andeven on the underlying mechanism, with a proposal that itmay also be autocatalytic (mediated by a ribozyme yet tobe discovered) instead of catalysed by a host RNAase, ashas been generally assumed.

Pathogenesis

Despite their structural simplicity, viroids may inducemacroscopic alterations comparable to those elicited bymore complex pathogens such as virus, bacteria or fungi.The symptomatology provoked by viroid infections can bequite variable. On leaves, symptoms include malforma-tions, epinasty, rugosity and chlorotic and/or necroticspots. Stems often show internode shortening, localized

necrosis on the vascular tissues and bark cracking.Deformations and colour alterations are frequent on fruitsand reserve organs, and delays in foliation, flowering andripening may also be observed. The phenotypical effectscan vary between different viroids, or between distinctisolates or hosts when considering the same viroid. Someviroids have devastating consequences, as CCCVd on thecoconut palm plantations of the Philippines, whereasothers incite very mild symptoms, as Grapevine yellowspeckle viroid(GYSVd)-1 or GYSVd-2 on grapevine, or nosymptoms at all, as Columnea latent viroid (CLVd) onColumnea. Viroid replication and symptom expression areusually favoured in plants grown at relatively high

temperature and light intensity. This is reflected by thefact that viroids affect mainly crops grown in tropical orsubtropical areas and in greenhouses. Infection by someviroids may lead to desirable traits on certain crops, asoccurs with citrus dwarfing. Thus, despite their usualnegative consequences, viroids may also have interestingagronomic applications.

Cytopathic and biochemical effects

The macroscopic symptoms observed in viroid-infectedplants are accompanied by alterations at other levels.Electron microscopy studies have shown the cytopathic

effects of viroid infection, which include distortion of cellwalls, accumulation of electron-dense deposits, appear-ance of membranous structures and disturbance of thechloroplast structure. Viroids trigger general defensivemechanisms of response to pathogens in the infectedplants. At the molecular level, effects of viroid infectionconsist of changes in the expression of proteins, like thepathogenesis-related (PR) proteins, and alteration in thelevels of hormones, like ethylene, or metabolites, likepolyamines.

Molecular models for pathogenesis

Both RNAs and proteins are potential candidates for thinitial target of viroid pathogenesis. Base-pair interactionbetween viroids and host RNAs, such as the small nucleaRNAs and the 7S RNA, might divert them from theinormal functioning and impair the cellular metabolism

Other host RNAs might also be affected if they araccidentally recognized and degraded by the hammerhearibozymes present in the Avsunviroidae members. On thother side, cellular proteins may also be a target for viroipathogenesis. Severe and mild isolates of PSTVd, CEVand CChMVd differ only in a few nucleotides (approxmately 1% of their sequences) but these changes may hava dramatic effect on the overall conformation or on domain of the viroid molecule, and alter its ability tinteract with one or more host proteins. In this respect correlation has been proposed between bending of the domain of PSTVd and symptom expression in tomato. Ihas also been shown that PSTVd is able to interact an

activate in vitro a mammalian 68-kDa protein kinase andinterestingly, the stimulation of a severe isolate is 10-folgreater than that of a mild one. This has led to thsuggestion that interaction of PSTVd with a planhomologue of this mammalian protein kinase, whosactivation triggers a cascade of reactions which ultimatelreduces initiation of protein synthesis, could be thprimary pathogenic effect of this viroid. However, interactions of the viroid RNA, or of its replicative intermediates, with host proteins involved in viroid replicationtransport or accumulation represent other possibilitieMoreover, as the two viroid families have very differenproperties, it is quite likely that there is more than on

pathway leading to pathogenesis.

Evolution

Viroids have a number of common structural features, liksize, circularity and lack of messenger activity, with somsatellite RNAs, which, in contrast to viroids, are functionally dependent on a helper RNA virus. Viroid-like satellitRNAs have self-cleaving ribozymatic domains in one o

both polarity strands which play a main role during threplication cycle via a rolling circle mechanism. Recently,new class of small plant RNAs have been describedrepresented so far only by the carnation small viroid-likRNA (CarSV RNA). Despite sharing structural similarities with viroid and viroid-like satellite RNAs, CarSVRNA is unique in lacking infectivity and in having a DNAcounterpart, an observation that led to the proposal thathe CarSV RNA and DNA are the two forms of retroviroid-like element.

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Viroid quasispecies

The complex genomic structure of viroid isolates reflectsthe extreme plasticity of these minimal pathogenic agents.Viroids do not propagate as uniform populations but as amixture of closely related variants fitting the quasispeciesmodel proposed for RNA replicons. The high sequence

heterogeneity of viroid isolates is essentially due to theaccumulation of mutants emerging de novo during replica-tion as a result of the error-prone nature of RNApolymerases. Although the frequent appearance of muta-tions is a main factor in viroid evolution, different selectionmechanisms seem to influence this process. Within thePospiviroidae family, preservation of some sequencemotifs, such as CCR, TCR and TCH, is presumablyrequired in viable mutants, together with the maintenanceof some other structural traits such as the rod-likesecondary structure and the possibility of adopting certainalternative metastable conformations with a potential roleduring replication (Qu et al., 1993). Within the Avsunvir-

oidae family, formation of hammerhead structures in bothpolarity strands seems critical for biological fitness.Another important factor limiting sequence heterogeneityin PLMVd and CChMV is the preservation of a branchedsecondary structure and, possibly, of a pseudoknot-likeinteraction in PLMVd. Besides the mentioned structuralconstraints, some sequence changes in viroids may also beconditioned by the hosts and tissues they infect, asillustrated by certain mutations only found in CEVdvariants isolated from citron or tomato, and by thesegregation of specific ASBVd sequences between sympto-matic and asymptomatic portions of the same leaf.

Studies on the molecular evolution of individual viroid

sequences have indicated that viroids can accumulatechanges rapidly. Although quick generation of newquasispecies after inoculation of plants with singlecomplementary deoxyribonucleic acid (cDNA) sequenceshas been reported for PSTVd and PLMVd, belonging todifferent families, the accumulation rate of sequenceheterogeneity is notably higher in the latter case. It hasbeen speculated that this could reflect the involvement ofdifferent RNA polymerases, with distinct mutation rates,in the replication of the two viroids.

Recombination in viroidsAlthough mutation seems to be the main source ofvariability of viroid evolution, RNA recombination alsoplays an important role. Evidence for genetic exchangesbetween viroids has been inferred from the quimericstructure of some viroids, like CLVd and Australiangrapevine viroid (AGVd), that suggests that they resultfrom recombination processes between several parentalsequences coinfecting the same host. Moreover, intramo-lecular rearrangements may also occur, as revealed from

the characterization of several CEVd and CCCVd variantcontaining sequence duplications.

Viroid origin

There are quite a few hypotheses about the origin oviroids, suggesting, for example, that they may come from

transposable elements or that they may represent escapedintrons. However, the most recent and attractive proposaassumes that viroid and viroid-like satellite RNAs may brelics of precellular evolution, in line with the idea of thexistence of a primitive RNA world composed of selfreplicating RNAs before the advent of DNA and proteinSeveral features of these RNAs support their consideratioas molecular fossils: small size, circularity (which woulpreclude the need of initiation and termination replicativsignals), high G1C content (which would attenuate theffects of the low fidelity of primitive polymerases) andmost remarkably, the catalytic nature of some of themAfter the evolution of cellular organisms, these free-livin

RNAs would have adopted an intracellular mode oexistence, with viroids and satellite RNAs becomindependent on the host and the helper virus, respectivelyPhylogenetic analyses of the sequences of viroid anviroid-like satellite RNAs are consistent with a commoorigin for both groups of RNAs.

Control

As for plant viruses, viroid control measures are essentiallprophylactic. Viroids are transmitted mainly by vegetativ

propagation and, consequently, the principal control is thuse of viroid-free propagating material. For this purposesimple and rapid methods for viroid detection are verimportant. Although biological tests with indicator planthave played an important role in the past, moleculamethods are now taking the lead. Since viroids do not codfor any protein, the enzyme-linked immunosorbent assay(ELISAs), which are based on antibodies raised againsviral proteins and have been broadly used for virudetection, are not applicable to viroids that must bdetected by other techniques which target the RNA itselProminent among these are molecular hybridization witradioactive and chemically-labelled cDNA- or cRNA

specific probes, and reverse transcription combined witthe polymerase chain reaction (RT-PCR) using specifiprimers. Most viroids are also transmitted mechanicalland the regular disinfection of pruning tools with dilutecommercial bleach is recommended. Some viroids, foexample ASBVd, are transmitted through seed and polleand this must be considered when establishing nurseriethat should be separated from infected areas. Only Tomatplanta macho viroid (TPMVd) is known to be efficientltransmitted by aphids under specific ecological condition

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in which naturally infected wild solanaceous plants arecultivated near tomato fields.

Elimination of viroids from infected plants

Some viroids such as PLMVd can be removed, althoughwith some difficulty, from infected peach trees by thermo-therapy (treating growing plants with hot air) followed bymeristem-tip propagation, but attempts to extend thisapproach to eliminate other viroids like Pear blister cankerviroid (PBCVd) have been unsuccessful. Since relativelyhigh temperatures are important for viroid accumulation,PSTVd has been eliminated by extended cold treatmentsfollowed by meristem-tip culture.

Biotechnological approaches for viroidcontrol

More recently, progress in molecular biology has opened

new windows for controlling viroid diseases. As alreadyindicated, the hammerhead structures found in threeviroids mediate self-cleavage in cis of the RNAs in whichthey are naturally embedded. But hammerhead structurescan also be engineered to act in trans, an aspect with majorbiotechnological implications. A good example of this isthe observation that transgenic potato plants expressing aribozyme against the minus PSTVd RNA display a highresistance against PSTVd replication, thus providing thefirst example of a ribozyme suppressing a viroid pathogento an undetectable level in planta (Yang et al., 1997).Control experiments showed that transgenic potato linesexpressing a catalytically inactive ribozyme were suscep-

tible to PSTVd. Inferior resistance levels were observed inplants transformed with ribozyme constructs targeting thePSTVd plus RNA, probably because viroid minus strandsaccumulate to lower concentrations and/or may be moreaccessible to the ribozyme. Since PSTVd replicates andaccumulates in the nuclei, where the transgenic constructscontaining the ribozymes are synthesized, these resultsagree with previous observations supporting the view thatthe effectiveness of ribozymes is dependent on colocaliza-tion with their target RNA substrates.

A second biotechnological approach for viroid controlhas been developed by expressing a double-stranded RNA(dsRNA)-specific RNAase from yeast in transgenic

potatoes. The introduction of the transgene, althoughnot causing anydeleterious effect in the growth pattern, didinduce an increased resistance to PSTVd infection in someof the transformed lines (Sano et al., 1997). The rationalebehind this strategy, which has also been applied toproduce virus resistant plants, is that the majority of plantviruses have single-stranded RNA genomes but replicatethrough intermediates containing dsRNA regions that canbe recognized and degraded by the dsRNA-specificRNAase. This mechanism may also operate against the

replicative intermediates of viroids, or against the viroimolecules themselves, that present a dsRNA-like structure. The potential of this approach is considerablbecause: the same construction may render a planresistant to a wide spectrum of viruses and viroids; thpathogen cannot escape by fixing mutations because thRNAase acts against the secondary structure and no

against sequence motifs; and no pathogen-derived sequences are released to the environment.

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Diener TO (1971) Potato spindle tuber virus IV. A replicating lo

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Grill LK and Semancik JS (1978) RNA sequences complementary t

citrus exocortisviroid in nuclei acidpreparations frominfectedGynur

aurantiaca. Proceedings of the National Academy of Sciences of th

USA 75: 896900.

Gross HJ, Domdey H, Lossow C et al. (1978) Nucleotide sequence ansecondary structure of potato spindle tuber viroid. Nature 273: 203

208.

Keese P and Symons RH (1985) Domains in viroids: evidence o

intermolecular RNA rearrangements and their contribution to viroi

evolution. Proceedings of theNationalAcademy of Sciences ofthe US

82: 45824586.

Navarro B and Flores R (1997) Chrysanthemum chlorotic mottle viroi

unusual structural properties of a subgroup of self-cleaving viroid

with hammerhead ribozymes. Proceedings of the National Academy o

Sciences of the USA 94: 1126211267.

Qu F, Heinrich C, Loss P et al. (1993) Multiple pathways of reversion

viroid conservation of structural domains. EMBO Journal12: 2129

2139.

Sano T, Candresse T, Hammond RW, DienerTO and Owens RA (199

Identification of multiple structural domains regulating viropathogenicity. Proceedings of the National Academy of Sciences o

the USA 89: 1010410108.

Sano T, Nagayama A, Ogawa T, Ishida I and Okada Y (199

Transgenic potato expressing a double-stranded RNA-specific ribo

nuclease is resistant to potato spindle tuber viroid. Nature Biotechno

ogy 15: 12901294.

Yang X, Yie Y, Zhu F et al. (1997) Ribozyme-mediated high resistanc

against potato spindle tuberviroid in transgenicpotatoes. Proceeding

of the National Academy of Sciences of the USA 94: 48614865.

Further Reading

Diener TO (1979) Viroids and Viroid Diseases. New York: Wiley.

Diener TO (ed.) (1987) The Viroids (The Viruses). New York: Plenum

Press.

Diener TO (1996) Origin and evolution of viroids and viroid-like satelli

RNAs. Virus Genes 11: 119131.

Ding B, Kwon MO, Hammond R and Owens R (1997) Cell-to-ce

movement of potato spindle tuber viroid. Plant Journal12: 931936

Flores R, Di Serio F and Herna ndez C (1997) Viroids: the non-codin

genomes. Seminars in Virology 8: 6573.

FloresR, Randles JW,Bar-Joseph M and DienerTO (1998)A propose

scheme for viroid classification and nomenclature. Archives

Virology 143: 623629.

Viroids



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Maramorosch K (ed.) Viroids and Satellites: Molecular Parasites at the

Frontier of Life. Boca Raton, FL: CRC Press.

Riesner D (1991) Viroids:from thermodynamics to cellularstructureand

function. Molecular PlantMicrobe Interactions 4: 122131.

Semancik JS (ed.) (1987) Viroids and Viroidlike Pathogens. Boca Rato

FL: CRC Press.

Symons RH (1997) Plant pathogenic RNAs and RNAcatalysis. Nucle

Acids Research 20: 26832689.

Viroids


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