VIROLOGY 177,2 16-224 (1990)

Structure, Self-Cleavage, and Replication of Two Viroid-like Satellite RNAs (Virusoids) of Subterranean Clover Mottle Virus’

CHRISTOPHER DAVIES,* JAMES HASELOFF,t AND ROBERT H. SYMONS’

Department of Biochemistry, University of Adelaide, South Australia 500 1, Australia; *The Sainsbury Laboratory, John lnnes Institute,

Colney Lane, Norwich NR4 7UH. United Kingdom; and tDepartment of Molecular Biology, Massachusetts General Hospital. Boston, Massachusetts 02114

Received January 3, 1990; accepted March 13, 1990

Both the genomic and viroid-like satellite RNAs (virusoids) from four subterranean clover mottle virus isolates de- scribed by Francki et a/. (1983, Plant Patho/. 32, 47-59) were analyzed in detail. Restriction endonuclease mapping of

cDNAs prepared from the genomic RNAs from all isolates showed that these RNAs are closely related if not identical. The two virusoids, which can occur together in the same isolate or individually, were sequenced and shown to be able to

form highly base-paired viroid-like secondary structures. The left-hand portions of these structures are almost entirely homologous but the right-hand portions show little similarity. The plus, but not the minus, virusoid RNAs contain se- quences that can form the hammerhead self-cleavage structure of certain other self-cleaving viroid, virusoid, and satel-

lite RNAs. Plus, but not minus, RNA transcripts from cDNA clones self-cleaved essentially to completion at the pre- dicted site during transcription in vitro. Northern blot analysis of infected leaf tissue extracts revealed the presence of

an oligomeric series of plus RNAs (of monomer size and greater) but minus RNAs were present only as high molecular weight species of heterogeneous size. These findings are in agreement with the lack of minus RNA self-cleavage in

vitro. Hence, these virusoid RNAs appear to replicate by a rolling-circle mechanism in which only the plus RNAs self-

cleave to form monomeric RNAs. o 1990Academic press, inc

INTRODUCTION

Subterranean clover mottle virus (SCMoV) (Francki et a/., 1983) is a member of a group of four closely related icosohedral plant viruses which encapsidate unique circular viroid-like satellite RNAs or virusoids. This group includes velvet tobacco mottle virus (VTMoV; Randles et al., 1981), solanum nodiflorum mottle virus (SNMV; Gould and Hatta, 1981), and lucerne transient streak virus (LTSV; Tien-Po et a/., 1981). These four vi- ruses are similar to Sobemoviruses (reviewed by Hull, 1988) in many respects but they differ in also contain- ing virusoids in field isolates (Francki, 1985, 1987; Keese and Symons, 1987).

The complete nucleotide sequences of the virusoids of VTMoV, SNMV (Haseloff and Symons, 1982), and LTSV (Keese et a/., 1983) are known. These RNAs are single-strand covalently closed circular molecules of 300-400 nucleotides and, like viroids, possess a high degree of intramolecular base-pairing. The sequences of two SCMoV virusoids are presented here.

Four isolates of SCMoV have been described (Fran- cki et a/., 1983). The virions from all isolates were found

’ Sequence data from this article have been deposited with the

EMBUGenBank Data Libraries under Accession Nos. M33000 and M33001.

’ To whom requests for reprints should be addressed.

to be of the same appearance as judged by electron microscopy and the coat proteins were indistinguish- able from each other by serological tests. All isolates contained a linear ssRNA of approximately 4.5 kb (the helper virus RNA). However, while the viral RNAs of the four isolates appeared to be similar, the virusoid com- ponents varied. Each isolate contained either one or both of two virusoids, termed ~(388) SCMoV and ~(332) SCMoV on the basis of their sizes as determined in this work. In the original work of Francki et al. (1983) these were referred to as 2 and 2’, respectively, based on electrophoretic mobilities.

In this paper we present evidence that there is a sin- gle virus which can support the replication of two different virusoids. The two vSCMoV RNAs are almost identical in sequence for roughly half of each molecule but little sequence similarity exists between the other halves. In addition, the in vitro self-cleavage via a ham- merhead structure of the plus RNA, but not the minus RNA, is shown. These findings are discussed in rela- tion to other self-cleaving RNAs and vSCMoV RNA rep- lication by a rolling circle mechanism.

MATERIALS AND METHODS

Virus and viral RNA purification

SCMoV isolates, kindly provided by Dr. R. I. B. Fran- cki (University of Adelaide), were mechanically inocu-

0042-6822190 $3.00 CopyrIght 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved

216

STRUCTURE AND SELF-CLEAVAGE OF VIRUSOIDS 217

lated to young clover (Trifolium subterraneum L. cv. Dinninup or cv. Mt. Barker) or pea plants (&urn sati- vum L. cv. Greenfeast). Two weeks postinoculation to- tal nucleic acids were extracted from leaf material (Re- zaian and Krake, 1987) or from purified virus (Francki et a/., 1983). Nucleic acids were also isolated from unin- fected leaf material by these two methods. Virus and virusoid RNAs were purified by polyacrylamide gel electrophoresis essentially as described by Gould (1981).

Synthesis and restriction endonuclease cleavage of SCMoV ds cDNA

Double-strand cDNA was produced from purified SCMoV RNAs by random priming, essentially as de- scribed by Gould and Symons (1982). These cDNAs were then digested with various restriction endonucle- ases and fractionated on a 6% polyacrylamide gel con- taining 2 M urea.

Sequence determination of vSCMoV RNAs

The nucleotide sequence of the virusoids from all four SCMoV isolates were determined using both RNA and cDNA sequencing methods. Linear RNA frag- ments of purified vSCMoV RNAs were generated by partial digestion under nondenaturing conditions es- sentially as described by Haseloff and Symons (1981). The resultant fragments were 32P-labeled at either the 5’- or 3’-ends, fractionated by polyacrylamide gel elec- trophoresis, eluted, and sequenced by the partial en- zymic cleavage method (Haseloff and Symons, 1981). The resultant RNA sequence was used to design two synthetic oligonucleotides which were complementary to regions conserved in both vSCMoV RNAs (5’-TTC- AAAATCCGCGAGGAGGG-3’ and 5’-CGAAAGTGA- GGTGGGGCC-3’). Double-strand cDNAs were synthe- sized from circular RNAs using these oligonucleotides as primers essentially as described by Gould and Sy- mons (1982). The ends of these DNAs were trimmed with Sl nuclease and ligated into bacteriophage M 13mpl8 and 19 DNA vectors. The resultant clones were then sequenced by the chain termination tech- nique (Sanger eta/., 1980). Overlapping sequences ob- tained from the RNA and cDNA sequence analysis were assembled to give the complete nucleotide se- quences of each vSCMoV RNA.

Cloning of vSCMoV cDNAs into RNA transcription vectors and RNA synthesis

A greater-than-unit-length 392 nucleotide cDNA clone (nt 294 to 21) of ~(332) SCMoV was prepared in pGem-2 (Promega) vector. The vSCMoV cDNA insert

was prepared by priming on purified circular RNA using synthetic oligonucleotide primers. 32P-labeled RNA probes for blot hybridization analysis were produced from this clone using eitherT7 RNA polymerase to pro- duce (-) transcripts or SP6 RNA polymerase to pro- duce (+) transcripts. The (-) and (+) transcripts are, respectively, of polarity opposite to or the same polarity as, the virusoid RNA.

A clone containing a subset of ~(332) SCMoV se- quences (nt 41 to 137) was prepared by digesting the greater-than-unit-length insert with Alul and Taql re- striction endonucleases and inserting the resultant 98 nucleotide fragment (after end-filling) into the Smal site of pGEM-1 vector.

Identification of the site of self-cleavage in ~(332) SCMoV RNA

The pGem-1 cDNA clone containing the AlullTaql fragment (see above) was linearized with HindIll and transcribed with T7 RNA polymerase in the presence of all four unlabeled NTPs. The (+) RNA transcripts pro- duced were purified on a denaturing polyacrylamide gel and the 109 nucleotide 3’-fragment resulting from self-cleavage was excised and eluted. This RNA was 32P-labeled at the 5’-end and sequenced by enzymic sequencing (Haseloff and Symons, 1981).

Northern blot hybridization analysis of vSCMoV RNAs

Nucleic acids were denatured with 1 M glyoxal (Mc- Master and Carmichael, 1977) in the absence of di- methyl sulfoxide and electrophoresed on 2% agarose gels in 10 mM sodium phosphate, pH 6.5. Nucleic acids were transferred to nitrocellulose by blotting for 16 hr and the filters were baked for 2 hr at 80” in vacua (Thomas, 1980). The filters were prehybridized, hybrid- ized, and washed as described by Thomas (1980) ex- cept for the following modifications. Filters were washed for 10 min at 90” in distilled water after baking (Hutchins et a/., 1985); prehybridization and hybridiza- tion were done at 55 and 68”, respectively, and after hybridization washing was carried out at 55”.

RESULTS

Four SCMoV isolates, A, B, D, and E, were described by Francki eta/. (1983). SCMoV-A contains both ~(388) SCMoV and ~(332) SCMoV RNAs with the helper virus RNA. In contrast, SCMoV-E contains only the helper virus RNA and ~(388) SCMoV RNA while SCMoV-B and SCMoV-D contain only the helper virus RNA and ~(332) SCMoV RNA. Interestingly, while isolates A, D, and E

218 DAVIES. HASELOFF. AND SYMONS

were obtained from the field, SCMoV-B was produced from SCMoV-A by passage through single lesions on pea leaves (Francki et a/., 1983). The helper virus and virusoid RNAs of all four isolates were analyzed in this work.

Analysis of SCMoV viral RNA sequences

In order to analyze the sequence relationships be- tween the viral genomic RNAs of the four isolates, ds- cDNAs were transcribed from purified RNAs using ran- dom oligonucleotide primers, and then digested with restriction endonucleasesA/ul, Haelll. Hhal, /-/pall, and Accl. Polyacrylamide gel electrophoresis of digested ds-cDNAs resulted in gel patterns which reflected the arrangement of restriction endonuclease recognition sequences in the original RNA molecules. As shown in Fig. 1 the cDNAs produced from all four isolated RNA species gave rise to indistinguishable patterns of restriction endonuclease fragments for the four nucleases shown. Similar results were obtained with Accl (results not shown). While the synthesized DNAs may not be wholly representative of the respective RNA species and small nucleotide sequence differences be- tween the RNAs may exist which do not affect the size or number of cDNA restriction endonuclease frag- ments, the results indicate that the viral RNA from the four isolates are certainly closely related in nucleotide sequence, and may be identical.

Sequence determination of ~(388) SCMoV and ~(332) SCMoV RNAs

The nucleotide sequences of the virusoid RNAs from all four isolates of SCMoV (Fig. 2) were determined us- ing both RNA and cDNA sequencing methods. No se- quence differences were observed between the ~(388) SCMoV RNAs obtained from SCMoV-A and SCMoV- E, or between the ~(332) SCMoV RNAs obtained from SCMoV-A, SCMoV-B, and SCMoV-D. The ~(388) SC- MoV and ~(332) SCMoV RNAs are 388 and 332 nucleo- tides in size, respectively. Secondary structure models for both RNAs (Fig. 2) show that they can base pair intramolecularly to form helical rod-like structures sim- ilar to those of the other virusoids and of the viroids (Keese and Symons, 1987). Alternative base pairings of the sequences around nucleotide 100 and other parts of both molecules are possible, only one of which is shown. Another base pairing arrangement has been presented in two reviews (Francki, 1987; Keese and Sy- mons, 1987).

The two vSCMoV RNAs share a region of approxi- mately 220 nucleotides of almost complete, contigu- ous sequence homology. Strikingly, the conserved

UNCUT Aiu I Hae III Hhal Hpa II

M lEASb inB= -Em

183,176 ry,

156 +

129,123 *

FIG. 1. Restriction endonuclease digests ds-cDNAs of SCMoVviral RNA. Double-strand cDNA was prepared from purified viral RNAs

from four SCMoV isolates, A, B, D, and E (Francki et al.,, 1983). The ds-cDNAs were either not digested or were digested with AU,

ffaelll, Hhal, or @all and fractionated on a 6% polyacrylamide, 2 M urea gel. The sizes (bp) of “P-end-labeled @all marker fragments of

M 13mp8 RF DNA (lane M) are indicated on the left-hand side.

sequences are located so as to form the entire base- paired left-hand portions of the proposed native struc- tures (Fig. 2). Consequently, it is the different lengths of the largely nonconserved right-hand portions of these molecules which account for their difference in size. The right-hand portions do contain some homologous sequences. The best match is 14 out of 18 nucleotides (shown by thick lines in Fig. 2).

Self-cleavage of ~(332) SCMoV RNA

The self-cleavage of ~(332) SCMoV RNA was investi- gated by using RNA transcripts produced by in vitro

219 STRUCTURE AND SELF-CLEAVAGE OF VIRUSOIDS

v(388)SCMoV

v(332)SCMoV

1

332 3i5 300 275 250 225 2bO

FIG. 2. Proposed secondary structures for ~(388) SCMoV and ~(332) SCMoV RNAs. Nucleotides conserved between both RNAs at similar

positions In the secondary structures are boxed. The site of self-cleavage is indicated by an arrow. An 18 nucleotide sequence of which 14

nucleotides are conserved in both RNAs is indicated by a thick line.

transcription of cDNAs in pGem-1 and pGem-2 vec- tors. Linearized pGEM clones containing ~(332) SC- MoV cDNA inserts both of partial- and of greater than unit-length were transcribed to give both (+) and (-) RNAs. The templates used for these transcriptions and the expected products are given in Fig. 3. The (+) but not the (-) RNAs underwent self-cleavage during tran- scription (Fig. 4). The fragments produced by self- cleavage were of sizes commensurate with the site of self-cleavage predicted from previously proposed sec- ondary structure models of other self-cleaving RNAs (Hutchins et al., 1986; Forster and Symons, 1987a,b; Keese and Symons, 1987; also see Discussion). En- zymic sequencing of the 5’.end of the (109 nt) 3’-self- cleavage fragment showed that the phosphodiester linkage between nucleotides C63 and U64 had been cleaved. Cleavage during transcription was so exten- sive that little or none of the uncleaved transcripts were visible (Fig. 4, lanes 3 and 5).

Northern blot hybridization analysis of vSCMoV RNAs from virions and plant extracts

In order to gain further understanding of the replica- tion of SCMoV RNAs and to assess the relevance of the RNA self-cleavage observed in vitro (Fig. 4) the na- ture of both (+) and (-) vSCMoV RNAs in virions and in infected plant cells was investigated. ~(332) SCMoV RNAs extracted from both virions and whole plant tis- sues infected with the SCMoV-D isolate were sub- jected to Northern blot hybridization analysis using RNA probes specific for either (+) or (-) sequences. Little or no hybridizing material was detected in any of the tracks in which nucleic acids from uninfected tis- sue had been run (Fig. 5, lanes 1, 3, 5, and 7). Probing

of RNAs extracted from purified virions for (+) vSCMoV RNA sequences revealed the presence of an oligo- merit series of bands (Fig. 5, lane 2). In long exposures a band corresponding to seven times the monomeric unit length was visible. In RNAs extracted directly from whole tissue only monomeric and a lesser amount of dimeric (+) RNAs were discernible together with some smearing (Fig. 5, lane 6); the monomer and dimer bands were clearly visible on the origin autoradiogram.

Minus RNAs were present at much lower levels than (+) RNAs. An extremely low level of high molecular weight (-) RNA was detected in RNA extracted from purified virions when probed for (-) vSCMoV RNA se- quences (Fig. 5, lane 4). Greater amounts of this high molecular weight (-) RNA were detected in total plant nucleic acid extracts (Fig. 5, lane 8); most of this hybrid- izing material was contained in a smear above an ill- defined band of approximately 1.3 kb. Monomer- or dimer-sized bands were not detected but a faint band which ran between the position of the (+) RNA mono- mer and dimer sized bands was present.

It is important to note that the film exposure time for the detection of (--) RNAs in lanes 3 and 4 of Fig. 5 was 50 times greater than for the detection of the (+) RNAs in lanes 1 and 2; the ratio was 9 times greater for lanes 7 and 8 as compared with lanes 5 and 6. The procedures used in the Northern hybridization analysis (Materials and Methods) are known to allow the detection of very low levels of (-) RNA in the presence of a large excess of (+) RNA of the same size (Hutchins et a/., 1985).

DISCUSSION Relationship between the SCMoV isolates

The four SCMoV isolates used in this work have been shown to be indistinguishable by serology and

220 DAVIES, HASELOFF, AND SYMONS

(a) Plus Hind III

(cl Plus c

Hind III

T7

(Hind Ill Hind Ill 1

C63-U64 6P6 RNA polymeraS%

t 5’ PA

/ I\ 294 332 1

m Hind III SF/SF I \

I

332 21

cleavage

I rQ

3’F (322ntl km I

5Ft126ntl

T7 RNA polymerase

I

I

\ 137 41

cleavage

LD I 3’F l109nt)

cI6;1 5’F 146ntl

(b) Minus (d) Minus EcoA I EcoR I

T7

VSCMOV v&

6F’6

Eco

I R l EC0 R I

T7 RNA polymerase sp6 RNA polymerase I

EcoR I 0 r//a 5’ 5’ er/la EcoR I

I I\ /C(164ntl\ 294 332 1

C (451nt) 33% \21 137 41

FIG. 3. Cloned ~(332) SCMoV cDNA templates and the RNAs resulting from their transcription with either SP6 or T7 RNA polymerases. (a) Greater than full-length ~(332) SCMoV pGem-2 template and the (+) RNAs produced by SP6 RNA polymerase transcription. (b) Greater than full-

length ~(332) SCMoV pGem-2 template and the (-) RNA produced byT7 RNA polymerase transcription. (c) Partial-length ~(332) SCMoV pGem- 1 template (containing theAlul/Taql fragment excised from the greater than full-length clone) and the (+) RNAs produced byT7 RNA polymerase transcription. (d) Partial-length ~(332) SCMoV pGem-1 template as in(c) but (-) RNAs were produced by transcription with SP6 RNA polymerase.

Hatched boxes, vector sequences; open boxes, vSCMoV sequences; solid boxes, SP6 and T7 RNA polymerase promoters. The self-cleavage sites with nt numbers are indicated by short arrows. For the (+) RNAs the complete transcripts are labeled 5’FB’F and the self-cleavage products 5’F and 3’F. For (-) RNAs the complete transcripts are labeled C and the (-) RNAs are numbered according to the (+) sequence.

electron microscopy (Francki et a/., 1983) and, in this work, by restriction endonuclease cleavage of cDNAs made to the viral RNAs. The virus isolates apparently differ therefore only in containing either one or both of the two virusoids. Further, there was no sequence vari- ation between the two isolates of v(388)SCMoV or be- tween the three isolates of v(332)SCMoV. However, it seems likely that sequence variants in the two different- sized virusoids would be found with sequence analysis of more isolates from different locations.

Sequence homology between 4388) SCMoV and ~(332) SCMoV

The predicted secondary structures of the two vSC- MoV RNAs show remarkable conservation of the left- hand portions of the molecules while the right-hand portions are almost entirely dissimilar (Fig. 2). Presum- ably this pattern has arisen from RNA recombination. Such intermolecular rearrangements have been pro- posed for viroids (Keese and Symons, 1985) a number

of other infectious plant RNAs (Haseloff et al., 1984; Bujarski and Kaesberg, 1986; Simon and Howell, 1986; Buzayan et al., 1987; Robinson et al., 1987; Kaper et a/., 1988) and plant viral defective interfering RNAs (Anzola et al., 1987; Hillman eta/., 1987).

It is probable that the common sequences and struc- tures in the vSCMoV RNAs mirror the apparent inter- changeable functions of these molecules. Thus the conserved left-hand portions may contain those se- quences required for replication by helpervirus or plant host components. Some of the conserved sequences contained in this region (Fig. 2) are shown to be respon- sible for the in vitro self-cleavage of ~(332) SCMoV RNAs (see below), a reaction considered to be an inte- gral part of the replication cycle. The constraints on the sequence of the largely nonconserved right-hand por- tions of these molecules are unknown but they pre- sumably must be able to form a base-paired structure and be within certain size limits. However, the limited homologies present in this region indicate the pres- ence of signals common to both RNAs. The sequenc-

STRUCTURE AND SELF-CLEAVAGE OF VIRUSOIDS 221

501,489 m

Lo4 -

FULL PARTIAL

nn

-+ -+

C

3’F

2 34 5

C

5’F

3’F

FIG. 4. Analysrs of the self-cleavage of v(332) SCMoV RNA. Autora- diograph of 5% polyacrylamide, 7 M urea gel showrng (+) and (-) RNA transcripts of greater than full-length and of partral-length ~(332)

SCMoV templates (Fig. 3). Lane 1, denatured 32P-labeled Hpall-di- gested pUCl9 DNA markers; sizes in nucleotides are given In the left-hand margin. Lane 2, menus RNA transcripts of greater than full-

length ~(332) SCMoV template. Lane 3, plus RNA transcripts of greater than full-length ~(332) SCMoV template. Lane 4. minus RNA

transcripts of partial-length ~(332) SCMoV template. Lane 5, plus RNA transcript of partial-length ~(332) SCMoV template.

ing of further vSCMoV isolates with a wider geographi- cal distribution would indicate if other variations to the right-hand regions are found or if only these two alter- natives exist.

Sequence homology between vSCMOV, vVTMOV, and vLTSV

The sequences and predicted secondary structures of vSCMOV, vVTMoV (Haseloff and Symons, 1982)

and vLTSV-A (Keese et a/., 1983) are shown in Fig. 6, and common sequences indicated. vSNMV is not shown due to its high degree of sequence homology with vVTMOV. There are a number of notable features. First, the sequence GAUUUU is present in all RNAs in a similar position on the secondary structures (approxi- mately located at residues 20-25 in all cases and indi- cated by a thick line in Fig. 6). The conservation of this sequence suggests that it may play some role which is common to the replication of these RNAs. Second, vSCMoV and vVTMoV RNAs share conserved se- quences (indicated by thin lines in Fig. 6) in addition to those directly involved in self-cleavage. Some of these sequences occur at the left-hand end of the central re-

Virion RNA Total RNA

SEQUENCES -

PROSED FOR + I+

x-75-7

--

UI UI

D

M

D

M

12 34 56 78

FIG. 5. Northern blot hybridization analysis of ~(332) SCMoV RNAs

extracted either directly from plant tissue or from purified virus. Nucleic acids from uninfected (U) and infected (I) fisum sativum L.

tissue were extracted either directly (total RNA) or from purified virus (vinon RNA). These nucleic acids were analyzed by Northern blot hy- bndrzation analysis using “P-labeled RNA probes specific for etther

(+) or (-) ~(332) SCMoV sequences. Bands corresponding to mono- meric (M) and dimeric (D) RNAs are indicated in the margins; tn lane 6, separate bands are clearly visible in the original autoradrogram.

Note that the monomeric and dimeric (+) RNAs from the total nucleic acids extract (lane 6) appear to have higher molecular werghts than those present in purified virus extracts (lane 2). This is because the

samples from total extracts contained viscous materials that slowed the migration of RNAs and marker dyes in the gel. Autoradiography was carried out as follows: lanes 1 and 2, 20 min; lanes 5 and 6, 2 hr. -80” with intensifying screen; lanes 3, 4. 7, and 8, 18 hr. -8O”, with intensifying screen.

324 300 275 250 225

G , I , u li

175 +

FIG. 6. Sequence and structural homology between v(388)SCMoV, ~(332) SCMoV, vVTMOV, and vLTSV-A RNAs. Due to the high degree of sequence homology between vSNMV and vVTMOV RNAs only the vVTMOV sequence has been presented. Residues common between all four

RNAs are boxed (these include those sequences that make up the hammerhead self-cleavage structures). The conserved GAUUUU sequence is highlighted by a thick line. Sequences conserved between vSCMoV and vVTMoV RNAs only are indicated by thin lines (some of these

sequences are those involved in the formation of the hammerhead structures but are not conserved in vLTSV). The self-cleavage sites of (+) and (-) RNAs are indicated by arrows

gion (to the right of those sequences involved in self- cleavage). These sequences may be involved in func- tions specific to these RNAs but they are unlikely to be essential for replication as LTSV can support the replication of vLTSV, vSCMOV, and vSNMV (Jones and Mayo, 1983, 1984; Jones et a/., 1983; Keese et al., 1983). Third, there are 13 nucleotides conserved be- tween all the RNAs (boxed in Figs. 6 and 7) that are involved in the self-cleavage of these RNAs in vitro (For- ster and Symons, 1987b; Symons, 1989).

sSCMoV RNA self-cleavage and replication

Plus-stranded transcripts from ~(332) SCMoV clones were shown to self-cleave in a site-specific manner during transcription in vitro. The RNA containing only a subset of ~(332) SCMoV sequences (nt 41-137)

cleaved as efficiently as the RNA containing the entire sequence, demonstrating that this sequence alone is sufficient for self-cleavage. This region in ~(332) SCMoV contains sequences conserved between the ~(332) SCMoV and ~(388) SCMoV RNAs that can be folded into hammerhead-shaped secondary structures (Fig. 7) similar to those proposed for the active self- cleavage structures of (+) and (-) avocado sunblotch viroid (ASBV), (+) and (-) vLTSV, (+) vVTMoV and vSNMV, and (+) satellite tobacco ringspot RNA (sTRSV) (Hutchins et a/., 1986; Keese and Symons, 1987; Forster and Symons, 1987a,b). The site of cleav- age predicted from this structure agrees with the dem- onstrated site of cleavage.

No cleavage of the (-) RNAs was observed and they do not contain a hammerhead self-cleavage domain. In addition, little sequence or structural similarity can be

STRUCTURE AND SELF-CLEAVAGE OF VIRUSOIDS 223

v(332)SCMoV

FIG. 7. Proposed single-hammerhead self-cleavage structure for

(+) ~(332) SCMoV RNA. The 13 boxed residues are those conserved in all RNAs that cleave by the hammerhead structure (Forster and

Symons, 1987b). The site of self-cleavage is lndlcated by an arrow. Base-paired stems are numbered I-III. The nucleotide marked with

an asterisk is a G in ~(332) SCMoV but an A in ~(388) SCMoV.

found between the (-) sTRSV RNA self-cleavage do- main (Buzayan et al., 1986) and the (-) SCMoV RNAs. It would therefore seem likely that the (-) SCMoV RNAs do not self-cleave, unless there is an as yet undiscov- ered self-cleavage mechanism that operates under conditions different from those used in this study. The proposed lack of cleavage of (-) SCMoV RNAs is sup- ported by the finding that the predominant (-) SCMoV RNAs detected in nucleic acids extracted from virions or directly from infected tissue are high molecular weight forms (Fig. 5). The problems associated with the detection of (-) RNAs in the presence of excess (+) RNAs have been adequately discussed by other work- ers (Branch and Robertson, 1984; Hutchins et al., 1985); we have followed the protocols of Hutchins et al. (1985) to alleviate these problems.

The cleavage of only vSCMoV (+) RNAs resembles the situation found in vVTMoV (and vSNMV) where the (+) but not the (-) sequences contain hammerhead self-cleavage domains (a small RNA containing the (+) vVTMoV self-cleavage domain has been shown to cleave in vitro; S. McNamara and R. H. Symons, unpub- lished results). Also, as for vSCMoV RNAs, only high molecular weight forms of (-) vVTMoV and vSNMV RNAs have been detected in plant tissue extracts (Chu et al., 1983; Hutchins et al., 1985). vSCMOV, vSNMV, and vVTMoV RNA replication can therefore be ex- plained by a rolling circle model in which transcription from a circular monomeric (+) RNA template gives rise to a greater than unit-length (-) RNA that does not un- dergo self-cleavage. This linear (-) RNA then acts as a template for (+) RNA synthesis. The (+) RNA is then cleaved and circularized, thus completing the cycle. This model is distinct from that which applies to sTRSV, vLTSV, and ASBV, where both (+) and (-) RNAs self-

cleave and where circular (-) RNAs act as templates for (+) RNA transcription and vice versa. It is also sim- ilar to the situation in plants infected with potato spin- dle tuber viroid and other related viroids where analo- gous evidence indicates only a single rolling circle dur- ing replication (Hutchins et al., 1985; Branch et a/., 1988).

ACKNOWLEDGMENTS

We thank Dr. Richard Francki for supplying the four Isolates of SC- MoV. helpful advice, and the use of glasshouse facilities, and Jennifer

Cassady and Tammy Greatrex for assistance. This work was sup- ported by the Australian Research Council and by a Commonwealth

Government Grant to the Adelaide University Centre for Gene Tech- nology.

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