J. theor. Biol.

(1984) 108, 519-527

IS the Conformation of Viroids Involved

in their Pathogenicity?
RICARDO FLORES

Instituto de Agroqufmica y Tecnologfa de Alimentos ( CSIC),

Jaime Roig 11, Valencia 10, Spain
(Received 30 June 1983, and in revised form 18 October 1983)
A model is presented that establishes a relationship between the pathogenicity of viroids and the conformation of a part of their molecules. The
model is applicable to the viroid group composed by potato spindle tuber
viroid, citrus exocortis viroid and chrysanthemum stunt viroid. It is assumed
that viroids adopt in vivo a rod-like structure which essentially coincides
with their so-called native structure in vitrp. The viroid pathogenicity is
associated with the conformation of a segment of the rod-like structure
between bases 43-44 and 54-56, and particularly with the alterations in
some specific points of the double-stranded structure of this segment. The
model is consistent with the observation that viroids of this group, with a
sequence homology of only 73%, induce in some hosts symptoms of similar
severity, whereas isolates of the same viroid, with a sequence homology
of 98% differ notably in their pathogenicity.

1. Introduction
Viroids are low-molecular-weight RNAs causing several diseases of higher
plants (Diener, 1979; Semancik, 1979). Important progress dealing with
viroid structure has been achieved, and the complete sequence o f some
members o f this new group o f infraviral pathogens has been elucidated
(S~inger, 1982). As regards viroid-host cell interactions the picture is much
less clear and, together with the origin of viroids and the mechanisms
involved in their replication, one of the most intriguing questions is how
viroids induce disease (Diener, 1982). As viroids are the smallest known
agents o f infectious diseases (Diener, 1974), they represent unique model
systems to study the processes that lead eventually to the appearance o f
characteristic symptoms in some of their hosts. The aim o f the present
communication is to discuss the hypotheses on viroid pathogenesis that
have been considered previously and, in the light of recent findings, to
present a model that establishes a relationship between the pathogenicity
of a group of viroids and the conformation of a segment of their molecules.
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O 1984 Academic Press Inc. (London) Ltd.

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2. Previous Models of Viroid Pathogenesis

All the explanations dealing with the mechanisms of viroid pathogenesis

have taken into account their lack of any detectable messenger function in
different translation systems (Davies, Kaesberg & Diener, 1974; Hall et al.,
1974; Semancik, Conejero & Gerhart, 1977). Other lines of evidence also
support this assumption (Diener, 1982). Therefore, it has to be concluded
that viroids exert their pathogenic effects by a direct interaction with a
cellular component, which results in an alteration of the normal gene
expression of the host cell. Several authors have suggested that viroids may
be a kind of regulatory RNA that has acquired an autonomous replication
(Diener, 1971; Semancik & Weathers, 1972; Robertson & Dickson, 1974;
Reanney, I975). In this respect, in the last few years two models have been
put forward as to how viroids could play a regulatory role.
First, from experiments in vivo (Miihlbach & S~inger, 1979) and in vitro
(Rackwitz, Rohde & S~inger, 1981 ; Flores & Semancik, 1982) it appears that
an enzyme with similar properties to DNA-dependent RNA polymerase II
is involved in viroid replication. Transcription assays with DNA-dependent
RNA polymerase II and the RNA of potato spindle tuber viroid (PSTV),
have revealed that this system is able to make full-length molecules of
complementary viroid RNA. This has been interpreted by considering that
viroids could compete with the genomic DNA for the template binding site
on the RNA polymerase II, altering in this way the synthesis of mRNA
(Rackwitz et al., 1981). Apart from the aforementioned transcription assays,
there is no additional experimental evidence in support of this hpyothesis.
It could be interesting to determine if a correlation exists between the
severity of the symptoms induced by several isolates of PSTV with different
base sequences (S~inger, 1982), and their affinity for RNA polymerase II,
as well as to investigate if the mRNA synthesis is impaired in some way in
viroid-infected tissues. Second, it has been proposed that viroids might have
originated by circularization of excised intervening sequences (introns)
(Diener, 1979; Crick, 1979). Moreover, from the comparison of viroid
sequences with the sequence of the small nuclear RNA UI (U1 snRNA),
which might have a key role in removing the introns from the precursor
molecules of eukaryotic mRNA (Lerner et al., 1980), a mechanism for viroid
pathogenesis has been formulated independently by several authors (Dickson, 1981; Diener, 1981; Gross et al., 1982). As their proposals differ in
some aspects it is worth to consider them separately.
In the first version (Dickson, 1981) two isolates of PSTV of known
sequences were considered (Gross et al., I978, 1981) causing symptoms of
intermediate (PD) and mild (PM) intensity; one Australian isolate of chry-

VIROID

CONFORMATION

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PATHOGENICITY

521

santhemum stunt viroid (CSV), whose primary structure had also been
established was also considered (Haseloff & Symons, 1981). According to
this hypothesis, viroids would adopt a conformation of intermediate stability
proposed previously (Riesner et al., 1979), which would permit them to
mimic the putative interactions of U1 snRNA with splice junctions of
eukaryotic mRNA precursors. The regions of the viroids analyzed for
potential base pairing with splice junctions were bases 306 to 314 and 112
to 123 (PSTV-PD), 305 to 313 and 112 to 123 (PSTV-PM) and 5 to 13 and
148 to 159 (CSV-Australia). These regions contain sequences homologous
to the 5' end of U1 snRNA. The only four differences found between the
primary structure of PSTV-PD and PSTV-PM lie within the regions of
proposed interactions with mRNA precursors. Moreover, as a result of these
base changes PSTV-PM would be expected to be a less effective competitor
for U1 snRNA than would PSTV-PD, a suggestion consistent with the
reduced severity of the symptoms induced by PSTV-PM (Dickson, 1981).
Recently, the sequence of a third isolate of PSTV inducing severe symptoms
(PS) has become available (S/inger, 1982). A comparison between the sequences of PSTV-PD and PSTV-PS shows no changes between bases 306 to 314
and 112 to 123. Furthermore, the primary structure of an English isolate of
CSV (Hollings & Stone, 1973), differing from CSV-Australia in ten nucleotide residues and in the total number of nucleotide residues, has been
worked out (Gross et al., 1982). A comparison between the sequences of
both isolates shows no changes between bases 5 to 13 and 148 to 159,
although CSV-Australia appears to be a more severe isolate than CSVEngland (see below). Therefore, this hypothesis does not provide an explanation for the different severity of the symptoms induced by the different
PSTV and CSV isolates.
In the second formulation (Diener, 1981) it was pointed out that a region
of homology exists between the 5' end of U1 snRNA and bases 257 to 279
of PSTV-PD. As a result of this homology the viroid may interfere with
the splicing process presumably mediated by the U1 snRNA's plant
equivalent. It should be indicated that the proposed region of homology
suggested in this hypothesis is different from that of the previous one
(Dickson, 1981). Moreover, the changes in the PSTV sequence responsible
for the different symptoms induced by the PM and PS isolates of PSTV do
not occur in the viroid segment between bases 257 to 279 and therefore, it
can not be explained the different pathogenicity of the three PSTV isolates.
Finally, in a third version (Gross et al., 1982) the PSTV-PD, the Californian isolate of citrus exocortis viroid (CEV) (Semancik & Weathers, 1972)
and the English isolate of CSV (Hollings & Stone, 1973) were taken into
account. Sequences (e.g., ACCCG) similar to those of U1 snRNA supposed

522

R, F L O R E S

to be involved in the recognition of pre-mRNA, occur in several locations

of the viroids considered. A more extended homology with the 5' end of
UI snRNA was found between bases 258 and 282 of PSTV-PD. This region
also exists in CEV-California and CSV-England. But as in the two previous
hypotheses (Dickson, 1981; Diener, 1981), these viroid regions homologous
to UI snRNA, do not contain the sequences where are located the base
changes associated with the different symptom severity of the PSTV and
CSV isolates.
3. A Conformational Model for Viroid Pathogenesis

From the above discussion it appears that the hypotheses considering

that viroids could exert their pathogenic functions by competing with the
genomic DNA or the splice sites of the host, do not have sufficient experimental data in favor or leave some important questions unanswered. Therefore, some alternative views must be sought. It has been reported recently
(S~inger, 1982) that most of the sequence differences in the PM, PD and PS
isolates of PSTV occur around nucleotide residues 40 to 50 and 310 to 320.
These base changes seem to alter to some degree the secondary structure
of this segment of the PSTV molecule and therefore, this may indicate that
pathogenicity of PSTV is determined in some as yet unknown way by a
structural domain around the aforementioned nucleotide residues (S~inger,
1982). I would like here to point out that the CEV and CSV have an
equivalent structural domain and, based on this observation, to present a
model where the pathogenicity of PSTV, CEV and CSV is associated with
the conformation of a definite segment of the viroid molecule comprised
between bases 43-44 and 54-56 (for simplicity I will refer only to the bases
of the upper part of the molecule, although it should be understood that
the whole structure of the segment is taken into account) (Fig. 1).
In order to establish a relationship between the conformation of PSTV,
CEV and CSV isolates in the interval from bases 43 A.4 to 54-56, and their
pathogenicity, it is pertinent to consider the severity of the symptoms that
they induce in three common hosts, Gynura aurantiaca, Lycopersicon esculenturn and Chrysanthemum morifolium. In this respect there are numerous
discrepancies in the literature that emerge probably from differences in:
viroid isolates (more than one severe and mild isolates of the PSTV have
been reported), propagation hosts, bioassay cultivars, type of inocula (crude
homogenates of tissue in buffer, partially purified and concentrated preparations of nucleic acids), inoculation procedures, age of the plant at the
moment of inoculation and environmental conditions (temperature, light).
Therefore, there is a need for comparative studies of symptom expression

VIROID C O N F O R M A T I O N

AND P A T H O G E N I C I T Y

<:/?: '''''" ?(i)

C G 0 0 UCU U U C

523

PSTV-PM

u c G u U u c U O c U~s]

1]U U C G U U C A U U C

u u G U

(J

CA

0 tJ

(1)~G G C A G G A A A A G~(:)
O C fi ~ C A C ~J 0 C

(11)~(i C uAUAG C A A A(~ ( t"5)

1~ G ~. tJ U

C GA

U O c ~J U (~

OO

pSTV-PS
CEV-Catiforn,a
CEV-AustraIia

CSV-Austroho

csv-Englona

FIG. 1. Proposed conformation following the procedure of Tinoco et al. (1973), of the
segment between bases 43--44and 54-56 of several isolates of PSTV, CEV and CSV. The arabic
numerals into brackets indicate the number of unpaired bases in the upper and lower halves
of the loops located at both ends of the segment. The arabic numerals enclosed by a circumference refer to base designations according to the convention established for PSTV by Gross et
al. (1978). In this latter case, for simplicity, numerals have been assigned only to the upper
part of the segment.
induced by different viroid isolates in host plants that have been inoculated
and grown under the same experimental conditions.
In the m e a n t i m e only a few tentative conclusions can be drawn. First,
C E V - C a l i f o r n i a induces severe s y m p t o m s in the three hosts (Niblett et al.,
1978, 1980). Second, PSTV-PS induces severe s y m p t o m s in G. aurantiaca
(Singh, 1973) and L. esculentum (Niblett et al., 1978) and of intermediate
intensity in C. morifolium (Niblett et al., 1978). Third, P S T V - P D , the " t y p e
strain" of this viroid, is assumed to be an isolate of intermediate severity,
considering the s y m p t o m s induced in L. esculentum (S~inger, 1982; Dickson
et al., 1977). Fourth, P S T V - P M induces mild s y m p t o m s in L. esculentum
and C. morifolium (Niblett et al., 1978) and no s y m p t o m s in G. aurantiaca
although this host is susceptible (Niblett et al., 1980). Fifth, b o t h C S V England and CSV-Australia induce s y m p t o m s in C. morifolium (Hollings
& Stone, 1973; Palukaitis & Symons, 1980) although only the latter can

524

R. F L O R E S

infect G. aurantiaca where it induces symptoms similar but less severe than
those induced by CEV-Australia (Palukaitis & Symons, 1980). Finally, the
sequence of one more isolate (obtained from S o l a n u m commersonii in
Scotland) has been determined very recently from molecular cloning experiments (Van Wezenbeck et al., 1982). The authors consider this PSTVScotland along with the PSTV-PD as severe isolates of this viroid. As
PSTV-PD appears now to be an isolate of intermediate severity this may
also be the case of PSTV-Scotland.
Figure 1 shows the conformation between nucleotide residues 43--44 and
54-56 of the several isolates of PSTV, CEV and CSV whose sequences are
known. It can be observed that between the right and left loops that limit
this segment of the viroid molecule, there are one or two central loops and
that when two central loops exist they are in all cases separated by two
base pairs. A correlation can be established between the amplitude and
number of these central loops and the severity of the symptoms induced.
Considering the PSTV isolates, the PM (mild) has only a 0 loop (the arabic
numerals indicate the number of bases in the upper and lower halves of
the loop) which is replaced in the Scotland and PD isolates (intermediate)
by a I and a 2 loops respectively, whereas in the PS (severe) there are two
central 22 loops. This conformation of PSTV-PS is remarkably similar to
that of CEV-California which, as has been mentioned before, also induces
severe symptoms in their hosts. According to this model, as CEV-California
and CEV-Australia have the same sequence in the segment of the molecule
which is being considered (Gross et al., 1982; Visvader et al., 1982), both
isolates should induce symptoms of similar severity, an assumption which
is supported by the few data in the literature about the biological properties
of CEV-Australia (Palukaitis & Symons, 1980). As regards the CSV, an
important part of the sequence ditterences between the two isolates are
located in the segment between bases 43 and 54-55 (Gross et al., 1982;
Haselott & Symons, 1981) and moreover, these differences alter notably the
conformation of this segment which has two central loops although of a
lower amplitude than those of PSTV-PS and CEV (Fig. 1). The CSVEngland, which is the less severe isolate of this viroid has, going from fight
to left, a o and a ~ loops, whereas the CSV-Australia, the more severe isolate,
has a ~ and a 0t loops. Therefore, it would appear as if the first loop should
be more important than the second one in determining pathogenicity.
Viroids are distinguished from other RNAs by a combination of three
mutually dependent features: circularity, an unusual secondary structure
and the dynamics of a highly cooperative structural transition (Gross &
Riesner, 1980). The alteration of the helix content of the segment between

VIROID

CONFORMATION

AND PATHOGENICITY

525

bases 43-44 and 54-56 should probably affect to some extent the mechanism
of denaturation of the viroid molecule, although the hairpins occurring in
intermediate structures during thermal denaturation of PSTV, CEV and
CSV (Gross et al., 1982) appear to remain essentially unchanged. Nevertheless it is altogether unclear whether the unusual dynamics of the structural
transition are important in viroid replication (Gross & Riesner, 1980) and
also very ditficult to imagine whether this might have some effect on
pathogenesis.
It has been stressed before (Diener, 1982), that any hypothesis of viroid
pathogenesis must be able not only to account for the symptoms of viroid
infection but also for the fact that in some plants, viroids are replicated
without detectable damage to the host (Diener, 1979). In the model discussed
here it can be conjectured that, because of the specific conformation of the
different viroid isolates between bases 43-44 and 54-56, they could interact
in a different way (or do not interact at all) with a cellular component. This
interaction would be responsible for the pathological consequences of viroid
infection. On even more speculative grounds, a protein might be considered
as a candidate for this cellular component since, as it has been noted
previously (McClement & Kaesberg, 1977), the hairpin-like structure of
PSTV may be related to viroid pathogenicity by providing the proper
conformation for specific interaction of the RNA with a protein. This idea
is supported by data from one of the best characterized systems to study a
sequence-specific RNA-protein interaction, that of phage R17 coat protein
and its RNA binding site for translational repression. It has been proposed
that coat protein recognition, probably requires some aspect of the secondary
structure of a fragment of the RNA molecule (the presence of a hairpin)
rather than simply a single-strand sequence (Gralla, Steitz & Crothers, 1974).
As a result of the formation of the RNA-protein complex, the melting
temperature of the hairpin is not altered although the rate of melting is
slowed. Further nuclease protection and selection experiments have defined
the binding site to a hairpin formed by 20 contiguous nucleotide residues
(Carey et al., 1983). Observations from other systems also lend credence to
this type of assumption. This is the case of the interaction between the
ribosomal 5S RNA and L18 protein, where a bulged nucleotide in an helix
fragment of the nucleic acid may provide a recognition signal for the protein
(Garret, Douthwaite & No ller, 1981). The specificity of these protein-nucleic
acid interactions appear to be quite high, giving support to the hypothesis
that a rather complex binding site on the RNA, with a particular threedimensional array of phosphates and nucleoside functional groups, is
needed for protein binding.

526

R. FLORES

4. Concluding Remarks
In summary, the model presented here to correlate the pathogenicity of
viroids with their conformation has the following features. First, only isolates
of PSTV, CEV and CSV are considered which are regarded as a separate
viroid group taking into account their sequence homology (Gross et al.,
1982; Haseloff & Symons, 1981; Visvader et al., 1982) and some of their
biological properties, particularly cross-protection (Niblett et al. 1978).
Second, it is postulated that viroids have in vivo and in vitro essentially the
same secondary structure, consisting of an arrangement of double-stranded
stretches separated by segments of unpaired bases in loops. This rod-like
structure has been obtained from the viroid sequence by minimizing the
free energy of helix formation according to an established procedure
(Tinoco, Uhlenbeck & Levine, 1973). It should be emphasized that although
there is biochemical and physicochemical evidence in support of this rodlike structure in vitro (S/inger, 1982), no experimental evidence exist indicating that viroids have the same structure in vivo. Third, viroid pathogenicity
is associated with the conformation of the left side of the rod-like structure
and particularly with the presence of specific loops interrupting the doublestranded structure of the segment between bases 43-44 and 54-56. Therefore,
the same segment is responsible for pathogenesis in all the viroid isolates
considered.
From the above paragraph it can be concluded, that the present model
establishes a relationship between the pathogenicity of a group of viroids
and their conformation, although it does not provide a detailed mechanism
for viroid pathogenesis. Nevertheless, I would like to stress that in this
model the molecular conformation, that has been considered repeatedly as
a very characteristic structural property of viroids, acquires an additional
meaning from the biological point of view. Moreover, the model is able to
explain the observation that isolates of different viroids, as PSTV-PS and
CEV-California, with a sequence homology of only about 73%, induce
very similar symptoms in some hosts, whereas two isolates of the same
viroid, as PSTV-PM and PSTV-PS, with a sequence homology of 98%,
differ remarkably in their pathogenicity. The elucidation of the sequence
of more isolates of this viroid group will help to determine if this model is
consistent. If this is the case, regions with a specific conformation responsible
for pathogenicity may also exist in other viroid groups.

I want to acknowledge the support of the Comisi6n Asesora de Investigaci6n

Cientifica y Trcnica of Spain.

VIROID CONFORMATION

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