Viroids: The Noncoding Genomes

Viroids: The Noncoding Genomes

Ricardo Flores,1 Francesco Di Serio, and Carmen HernandezInstituto de Biologia Molecular y Celular de Plantas (UPV-CSIC), Universidad Politecnica deValencia, Camino de Vera 14, 46022 Valencia, Spain

Viroids are independently replicating small circular RNAs which apparently do not code for proteins.They code, in a broad sense, for a conformation which is recognized and replicated by the host cell;two viroids also express ribozyme activities which probably mediate self-cleavage of the oligomericreplicative intermediates generated by a rolling circle mechanism. Viroids are classified into sub-groups according to their sequence and to the presence and type of some conserved motifs. Viroidinfections which induce symptoms, do so as a result of the direct interaction of the viroid itself, or aproduct of its replication, with a cellular target(s) of unknown nature. r 1997 Academic Press

KEY WORDS: hammerhead ribozymes; self-cleaving RNAs; rolling-circle replication; viroids; viroid-likesatellite RNAs.

INTRODUCTION

Twenty-five years after their discovery, viroids arestill the only subviral pathogens endowedwith autono-mous replication when inoculated to their hosts andwhich are well characterized at the molecular level.They were discovered as a consequence of studiesaimed at characterizing the agents of some plantdiseases initially thought to be induced by viruses.Different viroids have been identified as the etiologicagents of a series of maladies affecting both monocoty-ledonous and dicotyledoneous plants of economicimportance. Viroids consist only of a circular RNA of246–375 nt, a size significantly smaller than the ge-nome of the smallest virus. Differences between vi-ruses and viroids are not restricted to size because theavailable data support the conclusion that viroids, asopposed to viruses, do not code for protein. Therefore,viroids must rely on preexisting host enzymes for theirreplication and elicit their pathogenic effects by directinteraction between either the viroid RNA itself orother viroid-specific RNAs generated in the course ofthe infection and one or more cellular targets. Thisreview will focus on recent progress in understandingthe structure and function of these minimal agents.

Other aspects of viroid research have been discussed inprevious reviews (1–6).

PRIMARY STRUCTURE ANDCONSERVED SEQUENCE MOTIFS

Twenty-five members of the group (Table 1), andnumerous variants thereof, have been sequenced. Allknown viroids, except ASBVd and PLMVd, sharesome structural traits, in particular the presence of acentral conserved region (CCR) and lack a self-cleaving domains (see below). The CCR, located in thecentral domain of the rod-like secondary structureproposed to be adopted by most viroids in vitro (7,8),contains two sets of conserved nucleotides flanked byan inverted repeat in the upper strand (Fig. 1). Accord-ing to the core nucleotides there are basically threetypes of CCRs exemplified by those of PSTVd, ASSVd,and CbVd1; several subclasses can be distinguishedwithin the PSTVd CCR depending on the length of theconserved sequences (Fig. 1). The presence and type ofCCR are useful criteria for viroid classification (9),which leads to essentially the same grouping as thephylogenetic trees obtained by comparing the wholesequences (10).Another conserved sequence motif is the terminal

conserved region (TCR) (11), which is found in all1To whom correspondence and reprint requests should be ad-

dressed. Fax: 34-6-3877861.

Seminars in VIROLOGY 8, 65–73 (1997)

Article No. VI970107

1044-5773/97 $25.00Copyright r 1997 by Academic PressAll rights of reproduction in any form reserved. 65

members of the PSTVd and ASSVd subgroups and inthe two largest members, CbVd2 and CbVd3, of theCbVd1 subgroup (12,13). The presence of the TCR inthree different viroid subgroups, the strict conserva-tion of most of its sequence, CNNGNGGUUCCU-GUGG, and its similar location in the upper strand ofthe left terminal domain (Fig. 1) strongly suggest thatthis motif is involved in some critical function, prob-ably in replication. The lack of the TCR in the 248-ntCbVd1, as well as in the smallest members of thegroup (see below), suggests that this element existsonly in those viroids with a size greater than approxi-mately 300 nt.HSVd and the viroids of the CCCVd subgroup

(Table 1), all with a size of around 300 nt or smaller, donot have the TCR. However, the sequence CCCCU-CUGGGGAA found initially in HSVd, HLVd, andCCCVd (14) is also present in the two other membersof the CCCVd subgroup and forms a left terminalconserved hairpin (TCH) (Fig. 1). The conservation ofthis element in sequence, secondary structure, andlocation also favors an important functional role for it.ASBVd and PLMVd differ remarkably from the

other viroids: they lack a CCR and their RNAs of bothpolarities self-cleave because they contain hammer-head ribozymes (15,16).

HIGHER ORDER STRUCTURE LEVELS

Data obtained by different experimental approachesfavor the hypothesis that PSTVd adopts a rod-likesecondary structure in vitro, with short double-stranded regions separated by small single-strandedloops (7,8). However, recent results suggest the exis-tence of two small hairpins instead of an unbranchedstructure in the left terminus of most non-self-cleavingviroids as well as in ASBVd (17), and in the case ofPLMVd the whole structure of lowest free energy andother energetically close conformations is branched(16). Therefore, the universality of the rod-like struc-ture of viroids in vitro is doubtful. The structure ofviroids in vivo is unknown, although there is indirectsupport for at least a partial rod-like structure in someviroids, because the duplications or deletions observedin them preserve this type of structure (18–20).On the basis of sequence similarities, it has been

proposed that the rod-like structure of non-self-cleaving viroids contains five structural domains: C(central), P (pathogenic), V (variable), TL (terminalleft), and TR (terminal right) (Fig. 1) (21). These struc-tural domains were initially presumed to have specificfunctional roles; for example, the P domain was associ-ated with pathogenicity in PSTVd and related viroids,but the situation is now more complex and symptomexpression is thought to be controlled by discretedeterminants located within the TL, P, V, and TRdomains (22).In addition to the rod-like conformation, viroids can

also adopt metastable secondary structures containinghairpins (5). Hairpin I must be functionally relevantbecause it can be formed by all non-self-cleavingviroids and involves the core nucleotides of the CCRupper strand and the flanking inverted repeat (Fig. 2).The compensatory mutations preserving hairpin Iunderline the importance of some features of itsoverall structure, including the terminal tetraloop andthe adjacent 3-bp stem as well as the long stem at thebase, but there are also some conserved nucleotides(Fig. 2). The core nucleotides of the CCR upper strandand the flanking inverted repeat can form an alterna-tive palindromic structure which has been proposed tobe involved in processing of oligomeric viroid interme-diates (23,24). Hairpin II is formed by sequenceslocated in the lower strand of the rod-like structure at

TABLE 1

Classification of Viroids Which Have Been Sequenced

Viroid Abbreviation Size (nt) Subgroup

Potato spindle tuber PSTVd 356, 359–360 PSTVdTomato planta macho TPMVd 360 PSTVdCitrus exocortis CEVd 370–375 PSTVdTomato apical stunt TASVd 360, 363 PSTVdChrysanthemum stunt CSVd 354, 356 PSTVdIresine IrVd 370 PSTVdColumnea latent CLVd 370, 372–373 PSTVdHop stunt HSVd 297–303 HSVdCoconut cadang-cadang CCCVd 246–247 CCCVdCoconut tinangaja CTiVd 254 CCCVdHop latent HLVd 256 CCCVdCitrus IV CVd-IV 284 CCCVdApple scar skin ASSVd 329–330 ASSVdCitrus III CVd-III 294, 297 ASSVdApple dimple fruit ADFVd 306 ASSVdGrapevine yellowspeckle 1 GYSVd-1 366–368 ASSVd

Grapevine yellowspeckle 2 GYSVd-2 363 ASSVd

Citrus bent leaf CBLVd 318 ASSVdPear blister canker PBCVd 315–316 ASSVdAustralian grapevine AGVd 369 ASSVdColeus blumei 1 CbVd-1 248, 251 CbVd-1Coleus blumei 2 CbVd-2 301 CbVd-1Coleus blumei 3 CbVd-3 361–362, 364 CbVd-1Avocado sunblotch ASBVd 246–250 ASBVdPeach latent mosaic PLMVd 336–339 ASBVd

66 Flores, Di Serio, and Hernandez

Copyright r 1997 by Academic Press

both sides of the CCR, and its functional importancecan be inferred from two observations: it is conservedin all members of the PSTVd subgroup, and themaintenance of the core region is critical for PSTVdinfectivity (25).Elements of tertiary structure also exist in viroids.

An interaction of this class identified in PSTVd by UVirradiation (26) as well as by dimethyl sulfate modifica-tion (17), is particularly significant because it is also

present in loop E of 5S rRNA. This loop, containingnon-Watson–Crick basepairs, is part of the binding siteof at least two proteins: transcription factor IIIA, apositive regulator of 5S rRNA gene transcription, andribosomal protein L5, which has been implicated indelivering 5S rRNA to the nucleolus from the nucleo-plasm where it is transcribed. It would be tempting tospeculate that loop E could also act in PSTVd as abinding site for recruiting proteins involved in its

FIG. 1. Rod-like structure models for the different subgroups of non-self-cleaving viroids. The type member of each subgroup is indicated tothe right and the approximate locations of the five domains C (central), P (pathogenic), V (variable), and TL (terminal left), and TR (terminal right)(21), are on the top of the figure. The core nucleotides of the central conserved region (CCR), terminal conserved region (TCR), and terminalconserved hairpin (TCH) are shown. Arrows indicate flanking sequences which form, together with the core nucleotides of the CCR upperstrand, imperfect inverted repeats. The core nucleotides of the HSVd CCR have been defined by comparison with CLVd, which on the basis ofother criteria (the presence of the TCR, the absence of the TCH, and overall sequence similarity) is included in the PSTVd subgroup.Substitutions found in the CCR and TCR of IrVd, a newmember of the PSTVd subgroup, are indicated in lowercase. In the CbVd 1 subgroup theTCR exists only in the two largest members CbVd2 and 3.

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replication and/or intranuclear transport. The se-quences forming loop E are part of the CCR of thePSTVd and CCCVd subgroups and, therefore, thisloop can be formed quite probably by all members ofthese two subgroups. On the other hand, ASBVd andPLMVd must be able to adopt conformations withregions of tertiary structure because interactions of thistype, which are critical for the catalytic activity, havebeen identified in hammerhead structures.

REPLICATION: THE QUESTIONOF THE TEMPLATES

The subcellular location of some viroids has beenreexamined recently by in situ hybridization, confocallaser scanning, and transmission electron microscopy.In preparations of purified nuclei, PSTVd was locatedin nucleoli (27). In thin sections of tissue, CCCVd andCEVd were also located in nuclei, with the interestingdifference that CCCVd, but not CEVd, had accumu-lated to higher concentrations in the nucleolus than inthe nucleoplasm (28). Conversely, ASBVd has beenfound mainly in chloroplasts (29,30). If future workshows that PLMVd is similarly located, this willestablish a fundamental difference between the twomajor groups of viroids with implications for theirreplication and their evolutionary origin.Much evidence indicates that viroid replication oc-

curs by a rolling-circle model, with two variants andwith only RNA intermediates (Fig. 3) (31–34). In theasymmetric pathway, the infecting circular monomer

(which is assigned plus polarity by convention) istranscribed into linearmultimericminus strands, whichthen serve as the template for the synthesis of linearmultimeric plus strands. In the symmetric pathway,the linear multimeric minus strands are processed andligated to the minus circular monomer, which acts asthe template for the generation of the linear multimericplus strands. Therefore, one or two rolling circlesoperate in the asymmetric or symmetric variants,respectively. In both, the multimeric plus strands arecleaved and ligated to the plus monomeric circularRNA, the final product of the cycle. The distinctiveRNA of the symmetric pathway is the minus circularmonomer. Despite searching, this species has not beenfound in plants infected by PSTVd, which is thereforepresumed to replicate by the asymmetric pathway(33). The same pathway is also supposed to operate inthe replication of all non-self-cleaving viroids. Con-versely, preliminary indications of the existence of theminus circularmonomer in avocado infected byASBVd(32), which has subsequently been unambiguouslyconfirmed (34), strongly suggest that this viroid, andprobably PLMVd, replicate by the symmetric pathway.

REPLICATION: THE QUESTIONOF THE ENZYMES

Three enzymatic activities are required in the rollingcircle model: an RNA-dependent RNApolymerase forgenerating the multimeric strands, an RNase for pro-cessing them to unit-length linear strands, and an RNAligase to produce the circular monomers. The nuclearRNA polymerase II, acting on an RNA template, isquite probably involved in the synthesis of non-self-cleaving viroids, such as HSVd, PSTVd, and CEVd(35–38), since their replication is inhibited by the lowconcentrations (nanomolar) of a-amanitin, which in-hibit this enzyme. The most compelling evidence isthat synthesis of PSTVd-specific RNAs and of bonafide-polymerase II transcripts is similarly inhibited bya-amanitin in the same in vitro system (38). However,sound inferences in this direction must be restricted tosynthesis of the plus strands (37). It is problematic toconclude whether this, or a different enzyme, catalyzesthe first step of the cycle because it is very difficult todetect synthesis of minus strands in the presence of anexcess of the plus strands because of self complementa-rity.These results indicate that the nucleus is the site of

synthesis and accumulation of the aforementioned

FIG. 2. Hairpin I structures that can be formed by the CCR upperstrand of the five subgroups of non-self-cleaving viroids. Outlinedfonts indicate conserved nucleotides in similar positions in all thestructures.



viroids and probably of all non-self-cleaving viroids.Conversely, replication of ASBVd, and particularly ofits plus strands, is insensitive to high levels of a-amani-tin (39). This observation leads to two possibilities:ASBVd is replicated in the nucleus by an a-amanitin-resistant RNA polymerase (polymerase I or anotherunknown enzyme) and is then translocated to thechloroplast or the viroid accumulates and also repli-cates in the chloroplast, because the chloroplastic RNApolymerase resembles bacterial polymerase in beingresistant to a-amanitin. It must be emphasized that allthe previous conclusions about the enzymology of

viroid RNA polymerization are based on sensitivity toa-amanitin. Therefore, it would be very interesting toconfirm them with additional data obtained using adifferent approach. Two other intriguing aspects in thisregard are how viroids can recruit for their replicationcellular RNApolymerases, which under normal physi-ological conditions transcribe DNA templates, andwhether viroid strands have specific sequences for theinitiation and/or termination of transcription. HairpinI is a good candidate to be part of the origin ofreplication and/or cleavage, because it resembles theorigin of replication of geminiviruses, which also

FIG. 3. Models for viroid replication (31–34). The plus polarity (solid lines) is assigned by convention to the most abundant infectious RNA,and the minus polarity (open lines) to its complementary strand. The alternative asymmetric and symmetric pathways involve one or tworolling circles, respectively. In the symmetric variant, cleavage of plus and minus multimeric strands is mediated by hammerhead ribozymes(RZ), which lead to linear monomeric RNAs with 58-hydroxyl and 28-38-cyclic phosphate termini. Arrowheads denote the cleavage sites. Thehammerhead structures that can be formed by ASBVd (15,50) and PLMVd RNAs (16) are shown to the right; conserved nucleotides are boxedand substitutions found in two PLMVd cDNAclones are indicated in lowercase. In the asymmetric variant, cleavage of plus multimeric strandsis mediated by a host factor (HF), which generates linear monomeric RNAs containing probably 58-hydroxyl and 28-38-cyclic phosphate termini.

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replicate by a rolling circle mechanism, and whichcontains a similar structural element whose loop maycontain the cleavage site for replication (40).The RNase and RNA ligase activities involved in

processing of multimers of non-self-cleaving viroidsare presumably host protein enzymes. A nuclear ex-tract from potato cells can process linear oligomericPSTVd RNAs in vitro into infectious monomeric circu-lar RNA (41). However, the fungal RNase T1 can alsoperform the same function (42), showing that a nonspe-cific endoribonuclease, by itself, can catalyze RNAcleavage and ligation. Modifications in the procedurefor preparing the extract have led to the identificationof a different processing activity able to generateinfectious monomeric circular PSTVd from a mono-meric transcript containing a 17-nt repetition of theupper CCR strand, only when this transcript adopts aspecific secondary structure (43). Although some ofthese observations suggest that a host RNase catalyzesboth steps, a possible alternative is that an RNaseactivity produces linear monomers with 58-hydroxyland 28,38-cyclic phosphate termini which are thencircularized by an RNA ligase, because RNA ligase canmediate the in vitro circularization of naturally occur-ring PSTVd linear monomers (44,45). However, thelocation of the processing site is unclear because somedata point to the upper strand of the CCR, but otherdata indicate the existence of alternative cleavage sitesin the lower strand (46). Furthermore, transcripts witha short repetition of only 4 nt of the upper strand of theCCR (47), or with the exact monomeric length (48), areinfectious, and recent work has shown that the basicrequirement for infectivity of a range of RNA tran-scripts from monomeric CEVd cDNA clones appearedto be the ability of the transcripts to form shortdouble-stranded regions of viroid and vector se-quences at the junction of the two termini (49). There-fore, infectivity is independent of any duplication ofviroid sequences as previous experiments seemed toindicate.

REPLICATION: THE INVOLVEMENTOF RIBOZYMES

The data discussed above strongly suggest the in-volvement of host enzymes in the processing of oligo-meric PSTVd RNAs which provide the structuralinformation but not the catalytic activity (41–43). Repli-cation is different for ASBVd and PLMVd, which canform hammerhead structures in each polarity strand(Fig. 3) (15,16). These ribozymes excise linear mono-

mers both in vitro and in vivo because (i) the 58 terminiof two sub- and supragenomic plus ASBVd RNAs andthe 58 terminus of one subgenomic minusASBVd RNAisolated from infected plants were identical to thoseproduced by in vitro self-cleavage reactions (34), and(ii) nucleotide substitutions in two PLMVd variants inthe regions of both hammerhead structures did notaffect their stabilities, either because theywere compen-satory when in helices II and III or because they werelocated in loops (Fig. 3) (16). The activity of hammer-head ribozymes must be finely tuned during thereplication cycle: in some stages they must be opera-tive while in others, particularly when the final pluscircular monomer is synthesized, they must be inac-tive. Both viroids follow different strategies for thispurpose. The hammerhead structure of the plus ASBVdstrand is thermodynamically unstable because it has ahelix III of only 2 bp closed by a loop of 3 nt (Fig. 3),and its formation is very much restricted in themonomeric RNA, preventing in this way its self-cleavage. However, oligomeric ASBVd RNAs can forma stable double hammerhead structure (Fig. 3) (50),which quite probably mediates their self-cleavage tounit-length strands. On the other hand, the conforma-tion of the lowest free energy of the plus PLMVdstrand does not contain the hammerhead structureand, therefore, this RNA is not expected to self-cleave;this is the conformation which presumably exists inthe circular monomer. During transcription, the struc-ture of lowest free energy must be lost transiently,giving the active conformation the opportunity to formand promote RNA self-cleavage before synthesis hasbeen completed and the most stable conformationregained (16).It has been proposed that the final ligation step may

occur in PLMVd replication without the participationof proteins because linear monomers of this viroid,resulting from the hammerhead-mediated self-cleav-age of longer-than-unit transcripts, can self-ligate invitro (51). However, since 70% of the phosphodiesterbonds produced by self-ligation are 28, 58 (51), it isdoubtful that this could be the pathway in vivo. Theinvolvement of a host RNA ligase appears as a moreprobable alternative because at least in one viroid-likesatellite RNA, the junction resulting from ligation ofthe two termini generated by hammerhead-mediatedself-cleavage contains a 28 phosphomonoester, 38–58phosphodiester group (52). Such a group would be theexpected signature of a host ligase but not of self-ligation. In summary, there appears to be an interplaybetween two conformations, one containing the ham-merhead structure and promoting self-cleavage and a



second one facilitating circularization catalyzed prob-ably by a cell RNA ligase. A similar scheme could alsoapply for the non-self-cleaving viroids, although inthis case both cleavage and ligation steps would bemediated by protein enzymes.

EXPRESSION OF PATHOGENICACTIVITY

Since viroids do not appear to act as mRNAs, theymust exert their pathogenic effects by direct interactionwith cellular constituents. This primary effect wouldlead to the onset of symptoms through a signaltransduction pathway having as chainlinks pathogen-esis-related proteins, hormones, and metabolites likepolyamines (53). Both nucleic acids and proteins havebeen invoked as the initial viroid target. Althoughbasepair interactions between the viroid molecule andsmall nuclear or cytoplasmic (7S) RNAs appeared asan attractive hypothesis (see ref 54 for review), it isdifficult to explain how changes of three or fournucleotides could convert a severe PSTVd variant intoa mild one, particularly considering that the changesdid not map in the regions proposed to basepair.However, these changes can have a profound effect onthe three-dimensional conformation of the viroid mol-ecule. Severe andmild PSTVd variants of the same sizecan be separated by polyacrylamide gel electrophore-sis (55), and comparisons of the optimum secondarystructures of several PSTVd variants point to majordifferences in the geometry of their pathogenicitydomains (56). This makes the possibility of a protein(s)recognizing and interacting differentially with thedifferent structures a more likely alternative. Thenature of this protein is unknown, although recentresults suggest that activation of a plant enzymehomologous to the mammalian 68-kDa protein kinase(P68) could be the triggering event in viroid pathogen-esis (54). Activation of P68 triggers a series of reactionswhich leads to a diminution in protein synthesisinitiation. The most interesting observation is thedifferent effects PSTVd variants have on P68 in vitro,because the activation by the severe variant was atleast 10-fold greater than that by the mild one (54).Alternatively, interaction with a transcription factorrequired for viroid replication, or with a proteininvolved in viroid transport and/or accumulation,could be the initial event in viroid pathogenesis.Probably there is more than one mechanism of viroidpathogenesis because ASBVd differs from PSTVd andclosely related viroids, in the RNA polymerase in-

volved in its replication (39) and in its site of accumula-tion (29,30) (see above).

INTERFERENCE BETWEENCO-INFECTING VIROIDS

Crossprotection is known to occur between strainsof the same viroid and between different viroids whichare very similar in sequence (57). In general, there is agood correlation between the delay in the onset ofsymptoms induced by the more severe member of apair of co-inoculated viroid RNAs and the delay in itsaccumulation (55,58,59). On the other hand, concur-rent inoculations with some viroid pairs resulted ininterference because one of the pair outcompeted theother (58,60,61). These data suggest that a limiting hostfactor is needed for viroid replication, transport, oraccumulation and that differential affinity for thisfactor determines which of the co-inoculated viroidsprevails. Irrespective of the mechanism of viroid cross-protection, it must be different from that of crossprotec-tion among viruses because of the very dissimilarnature of viroids and viruses. It is also possible thatthere is more than one mechanism of crossprotectionbetween viroids, because the phenomenon has beenobserved between pairs of both self-cleaving andnon-self-cleaving viroids.

ACKNOWLEDGMENTS

We thank Professor H.L. Sanger for making available to usunpublished results from his laboratory and Dr. V. Pallas for criticalreading of the text. Work from R.F. laboratory is supported by GrantsPB92-0038 and PB95-0139 from the Direccion General de Investiga-cion Cientifica y Tecnica de Espana and by contract CHRX-CT94-0635 from the European Union. F.D.S. and C.H. are recipients ofpostdoctoral fellowships from theMinisterio de Educacion y Cienciade Espana and the European Union, respectively.

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Viroids: The Noncoding Genomes