self-cleavage of rna in the replication of viroids …self-cleavage of viroid and virusoid rna 307 a...

y. Cell Sci. Suppl. 7, 303-318 (1987)Printed in Great Britain © The Company o f Biologists Limited 1987

303

SELF-CLEAVAGE OF RNA IN THE REPLICATION OF VIROIDS AND VIRUSOIDS

RO BERT H. SYM ONS, CH ERYL J. H U TC H IN S, ANTHONY C. FO R ST ER , PETER D. RA TH JEN , PAUL K E ESE a n d JA N E E. VISVADERDepartment of Biochemistry, University of Adelaide, Adelaide, South Australia, 5000

S U M M A R Y

Viroids are infectious, circular RNA molecules of 246 to 375 nucleotides found in plants. Virusoids are of similar size and structure but they are dependent on, and encapsidated in, a helper virus. A rolling circle mechanism of replication is considered to account for the presence of greater- than-unit-length plus and minus RNAs of both viroids and virusoids found in infected plants. An essential feature of this mechanism is the specific processing or cleavage of high molecular weight intermediates to produce linear monomers which are then ligated to circular monomers.

We have investigated the putative processing cleavage reactions using in vitro-synthesized RNA transcripts of dimeric cDNA clones of the 247-nucleotide avocado sunblotch viroid (ASBV) and of partial cDNA clones of the 324-nucleotide virusoid of lucerne transient streak virus (vLTSV). In both cases, there is a specific, non-enzymic, self-cleavage of plus as well as minus transcripts. The plus and minus sites of cleavage are in neighbouring parts of ASBV and of vLTSV and highly conserved two-dimensional structures can be drawn around the cleavage sites as well as around the putative or demonstrated cleavage sites of precursors of the virusoids of three other viruses and of the linear satellite RNA of tobacco ringspot virus. The results also indicate that the sole function of about one-third of the ASBV and vLTSV molecules is provision of sequences that allow the formation of the self-cleavage structures of both ‘plus’ and ‘minus’ RNA precursors during the replication cycle.

Similar self-cleavage of ‘plus’ RNA transcripts of a dimeric cDNA clone of citrus exocortis virus (CEV) was not observed. However, the putative processing site for CEV precursors was located within three nucleotides by site-directed mutagenesis. No two-dimensional structures similar to those found for ASBV and vLTSV were found around the processing site. It is possible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle of CEV and related viroids.

I N T R O D U C T I O N

Viroids, the smallest known pathogens of higher plants, are infectious, singlestranded, circular RNAs of between 246 and 375 nucleotides (Riesner & Gross, 1985; Keese & Symons, 1987). Although virusoids are similar to viroids in being singlestranded, circular RNAs of between 324 and 388 nucleotides, they comprise one of a number of groups of satellite RNAs which are dependent on a helper virus for infection (Murant & Mayo, 1982; Francki, 1985; Keese & Symons, 1987). Replication of viroids and virusoids does not involve a DNA intermediate and there is no evidence for viroid- or virusoid-coded polypeptides. Hence, they must rely on sequence and structural signals for their interaction with the host plant.

A common feature of the replication of viroids and virusoids is the presence in infected plants of greater-than-unit-length ‘plus’ (same sense as infectious RNAs) and complementary ‘minus’ RNAs (Branch & Robertson, 1984; Ishikawaei al. 1984;

304 R. H. Symons and others

Hutchins et al. 1985). A rolling circle mechanism of replication is considered to account for these RNAs. An essential feature of such a mechanism is the specific processing or cleavage of ‘plus’ and, sometimes, ‘minus’ high molecular weight intermediates to produce ‘plus’ or ‘minus’ linear monomers which can then be ligated to circular monomeric species.

We summarize here our work on the replication of the 247 nucleotide avocado sunblotch viroid (ASBV) and the 324 nucleotide virusoid of lucerne transient streak virus (vLTSV) which indicates that this processing is a non-enzymic self-cleavage reaction. An important finding of this work is that in vitro transcripts of ‘plus’ and ‘minus’ ASBV and vLTSV readily and specifically self-cleave and that highly conserved sequences and two-dimensional structures occur adjacent to the cleavage sites. Results so far obtained with the 371 nucleotide citrus exocortis viroids (CEV) indicate that CEV and related viroids make use of a different processing mechanism during the replication cycle.

M U L T I M E R I C P L U S A N D M I N U S F O R M S OF A S B V A N D OF TWO V I R U S O I D S

The approach we and others have used to determine the replication intermediates of viroids and virusoids was to prepare partially purified nucleic acid extracts of infected plants, denature the nucleic acids by treatment with glyoxal or formaldehyde followed by fractionation by agarose gel electrophoresis. After transfer of the nucleic acids to nylon membranes, they were hybridized with full-length singlestrand ‘plus’ and ‘minus’ 32P-DNA and RNA probes derived from recombinant DNA clones. The results obtained for ASBV, vLTSV and the 378 nucleotide virusoid of solanum nodiflorum mottle virus (vSNMV) are shown in Fig. 1 (Hutchins et al.1985). '

The pattern obtained for all ‘plus’ RNAs was an oligomeric series of RNAs; up to 10 bands could often be counted on the original autoradiograms. That these bands were integral multiples of the monomeric species was confirmed in each case by a linear plot of the mobility of each band against the logarithm of the putative Mr and by their sensitivities to pancreatic and T] RNases. In the case of ASBV, minor bands between the major bands were reproducibly found but have not been characterized (Bruening et al. 1982; Hutchins et al. 1985).

There was significant variation in the patterns of the ‘minus’ species (Fig. 1) which, however, always occurred at much lower levels than the ‘plus’ species. In the case of ASBV, only monomeric and dimeric ‘minus’ species were found. With vLTSV a monomeric ‘minus’ species was accompanied by a trace of high molecular weight material. In contrast, the ‘minus’ vSNMV RNAs consisted mostly of a broad range of high molecular weight species. A similar result for the virusoid of velvet tobacco mottle virus (vVTMoV) was reported by Chu et al. 1985).

Both ‘plus’ and ‘minus’ monomeric and dimeric ASBV consisted of circular and linear molecules when nucleic acid extracts were examined by polyacrylamide gel electrophoresis in the presence of 7 m urea (Fig. 2; Hutchins et al. 1985), conditions

ASBV LTSV VTMoV

Self-cleavage of viroid and virusoid RNA 305

+

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Fig. 1. Detection of ‘plus’ and ‘minus’ sequences of ASBV, vLTSV and vVTMoV in nucleic acid extracts of infected leaves. Glyoxalated nucleic acid extracts ‘plus’ markers were fractionated by electrophoresis in 1-9% agarose gels in 10 mM sodium phosphate, pH 6-0. Nucleic acids were bi-directionally transferred on to nylon membranes which were baked for 2-4 h in vacuo at 80 °C. Prehybridization and hybridization were carried out essentially as described by Thomas (1983). Single-stranded ‘plus’ and ‘minus’ 3ZP- cDNA probes which detect plus and minus sequences, respectively) were prepared as in Hutchins et al. (1985) and Barker et al. (1985). Autoradiography was carried out at room temperature for the detection of the plus species and at —70°C in the presence of intensifying screens for the detection of minus species. O, origin of gel; M, D, T, monomer, dimers and trimer species, XC, xylene cyanol FF marker dye. A plus monomer marker was used in each case (not shown). + and — tracks probed with ‘plus’ and ‘minus’ probes, respectively. Data from Hutchins et al. (1985).

known to cause the separation of circular and linear forms of viroids (Brueningei al. 1982; Palukaitis & Symons, 1980). The marker monomer and dimer ‘plus’ RNAs were obtained by elution from non-denaturing gels after staining followed by further purification on urea gels (Brueningei al. 1982). The presence of linear species in the circular markers illustrates the difficulty of purifying circular molecules without nicking. There was no hybridization of the ‘minus’ probe to these markers, indicating that the monomer and dimer ‘minus’ species did not co-electrophorese with the ‘plus’ species during the two gel purification steps. The ‘minus’ monomer of vLT SV has not been checked to see if it also contains circular molecules.


A Plus DNA probe B Minus DNA probe

DC-

D L -

x2-

MC-

1 2 3 4 5 6 7 8 9 10ML MC Ex DLDC+DL MC Ex DL DLDC+DL

Fig. 2. Circular and linear forms of ‘plus’ and ‘minus’ monomeric ASBV in nucleic acid extracts of infected avocado leaves. Heat denatured but non-glyoxalated nucleic acid extracts and marker monomer and dimer ASBV (Bruening et al. 1982) were fractionated by electrophoresis on a 4% polyacrylamide gel in 9 0 mM Tris-borate, 2mM EDTA, 7 m urea. Nucleic acids were transferred to a nylon membrane by electroblotting in 25 mM sodium phosphate, pi I 6-5 and probed with single-stranded ,2P-‘plus’ and ‘minus’ cDNA probes. Lanes 1 and 6, linear monomer ASBV (M L); lanes 2 and 7, circular monomer ASBV (M C); lanes 3 and 8, nucleic acid extracts of infected leaves monomer ASBV (M C); lanes 3 and 8, nucleic acid extracts of infected leaves (Ex); lanes 4 and 9, linear dimer ASBV (D L ); lanes 5 and 10, linear circular dimer of ASBV (DC + D L). Taken from Hutchins et al. (1985).

R O L L I N G C IR C L E M O D E L FOR R E P L IC A T IO N OF V IR O ID S A N D V I R U S O I D S

On the basis of results presented in F'ig. 1 and elsewhere (Bruening et al. 1982; Hutchins et al. 1985; Branch & Robertson, 1984), a rolling circle mode of replication (Fig. 3) is considered best to fit the data available. The model of Fig. 3(A) is applicable to ASBV and vLT SV . The infecting circular monomeric species is copied by a host RNA polymerase (yet to be identified) to produce an oligomeric ‘minus’ strand which is specifically cleaved to a linear monomer. This is then circularized by a host RNA ligase and acts as a template for an RNA polymerase to copy into an oligomeric ‘plus’ strand. Specific cleavage to the monomer and circularization gives the circular plus RNA which is the dominant form found in vivo.

The specific cleavage could occur by the action of a host RNase or by a non- enzymic mechanism similar to that described for Tetrahymena rRNA (Cech, 1986).

-xc


A B G )

42

Minus

3

Fig. 3. Two rolling circle models for the replication of viroids and virusoids (Branch & Robertson, 1984; Hutchins et al. 1985). (A) A circular input ‘plus’ strand is copied by a host RNA polymerase to give an oligomeric ‘minus’ strand which is processed at specific sites (squat arrows) to give a full-length minus strand. This is circularized by an RNA ligase and copied to give an oligomeric ‘plus’ strand w'hich is also processed to full-length linear ‘plus’ strands w'hich are circularized to give the progeny viroid. (B) Similar to mechanism A except that the oligomeric ‘minus’ strand is not processed but copied to give an oligomeric ‘plus’ strand.

As we show below, a much simpler self-cleavage reaction appears to be involved. The presence of oligomeric forms of viroid or virusoid can be explained by the inefficiency of the cleavage reaction such that the cleavage does not occur at every site and/or, by tandem ligation of linear monomers by the RNA ligase, with or without circularization.

The model in Pig. 3(B) is a variation on that in Fig. 3(A) (Branch & Robertson, 1984) and is consistent with the results obtained with vSNM V (Fig. 1). The oligomeric ‘minus’ strand is not cleaved but acts as a template for the non-rolling circle copying to produce an oligomeric plus strand which is cleaved to the linear ‘plus’ monomer. It cannot be excluded that both routes in Pig. 3(A) and 3(B) may occur to varying extents during the replication of some viroids and virusoids.

S E L F - C L E A V A G E OF N A T U R A L D I M E R S OF A S B V

During ‘Northern’ hybridization analysis of replication intermediates present in extracts of avocado leaves infected with ASBV (Figs 1,2) we noted that purified ASBV dimers used as markers spontaneously and specifically cleaved to linear monomers on long term storage at — 20°C and occasional freeze-thawing. This observation, coupled with evidence that purified linear dimeric and trimeric forms of the 359 nucleotide satellite RNA of tobacco ringspot virus (vTRSV ) (Buzayan et al. 1986) were also specifically cleaved to monomers (Prody el al. 1986), prompted us to investigate self-cleavage reactions in oligomeric ASBV.

Purified circular and linear dimers of ASBV (Bruening et al. 1982) when incubated under a variety of ionic conditions in the absence of protein, were converted to linear monomers (Fig. 4). Difficulties associated with the purification of sufficient quantities of dimers, the time consuming ‘Northern’ hybridization analysis


required to follow each reaction, inconsistencies in the extent of self-cleavage observed between different dimer preparations, and the impossibility of looking at ‘minus’ ASBV species, led us to in vitro transcription systems for the preparation of ‘plus’ and ‘minus’ ASBV.

1 2

Fig. 4. In vitro conversion of ASBV circular or linear dimers to linear monomers. A purified mixture of circular and linear ASBV dimers (Brueningei at. 1982) was incubated in 50 mM Tris-HCl, pi I 7-5, 10mMMgCl2, 100 mM (NH4)2S 0 4, 0-1% SD S, for lh at 37°C. Conversion of dimers (lane 1) to linear monomers (lane 2) was detected by ‘Northern’ hybridization analysis using single-stranded M13 phage DNA probes containing a full-length ASBV insert (Hutchins et at. 1985). Electrophoresis was on a 4% acrylamide, 7 M urea gel. Positions of markers run at the same time are shown. DC, dimer circles; D L, dimer linears; MC, monomer circles; ML, monomer linears.


S E L F - C L E A V A G E OF R N A D U R I N G I N V I T R O T R A N S C R I P T I O N OF c D N A

C L O N E S OF A S B V A N D v L T S V

Dimeric cDNA clones of ASBV and partial length clones (273 nucleotides) of vLTSV were prepared in pSP64 and pSP65 vectors containing the promoter for the phase SP6 RNA polymerase (Melton et al. 1984) (Fig. 5(A),(B)). Transcription of the Sm aI-linearized pSP64 dimer ASBV clone was expected to generate a full-length transcript C of 528 nucleotides. However, three major ‘plus’ products, M, 5'E and 3'E in addition to lesser amounts of the putative full-length dimer transcript C were observed when the reaction mixture was analysed on a denaturing polyacrylamide gel (Fig. 5(C), lane 2). Product M co-electrophoresed with a marker (ML, not shown) of purified linear monomer of 247 nucleotides (Symons, 1981) and indicated that an ASBV monomer was specifically cleaved from the dimer transcript.

The identity of the products smaller than complete transcript C was determined in two transcription reactions. Incorporation of label only into product 5'E and the full- length transcript C during transcription in the presence of [y-32P]GTP, in place of [ar-32P]GTP, established that these additional products were not the result of premature termination of transcription (Fig. 5(C) Lane 1). When non-linearized vector was transcribed in the presence of [cv-32P]GTP transcription could continue well beyond the ASBV sequence into the vector sequence. In this case (Fig. 5(C) lane 3), label was found only in products M and 5'E plus a smear of high molecular weight material. It was concluded that product 5'E was the terminal 5'-fragment and product 3'E the terminal 3'-fragment resulting from excision of the full-length linear ASBV monomer from complete transcript C (Fig. 5(A)). Cleavage at only one of the two possible cleavage sites was considered to give rise to minor products 5'P and 3'P (Fig. 5(C)).

Similar results were obtained on the SP6 RNA polymerase transcription of a pSP65 ASBV minus dimer clone linearized with//m dIII (not shown; see Hutchins et al. 1986). Thus, a complete transcript of 534 nucleotides cleaved to produce a 5'- terminal and a 3'-terminal fragment and a 247 nucleotide ‘minus’ linear monomer as well as partial products produced by cleavage at one of the two possible sites. In the case of both ‘plus’ and ‘minus’ species, the predicted sizes of all products was confirmed by the sizing of products by gel electrophoresis using single-strand DNA markers.

Similar experiments to those of ASBV were also performed with partial length (273 nucleotide) ‘plus’ and ‘minus’ clones of the 324 nucleotide vLTSV (Fig. 5(B), (D)). Transcription of the iscoRI-linearized ‘plus’ clone with SP6 RNA polymerase gave the expected full-length 336 nucleotide transcript C and the two cleavage fragments 3 'F and 5 'F (Fig. 5(D), lane 1). The non-linearized plasmid gave a smear of high molecular weight material and labelled fragment 5 'F but not 3 'F (Fig. 5(D), lane 2). Analogous results (not given) were obtained with the minus clone of vLTSV.


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TRANSCRIPTION SELF-CLEAVAGE

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Fig. 5.


C L E A V A G E OF R N A T R A N S C R I P T S O C C U R S I N T H E P R E S E N C E OF A D I V A L E N T

C A T I O N A N D I N T H E A B S E N C E OF P R O T E I N

Although specific cleavage of both ‘plus’ and ‘minus’ RNA transcripts of the ASBV and vLTSV clones occurred during transcription in the absence of plant proteins, it was necessary to determine if this could also occur in the absence of any protein. Hence, full-length transcript C of ‘plus’ (Fig. 5(C), Lane 2) and ‘minus’ ASBV and transcript C of ‘plus’ (Fig. 5(D), lane 1) and ‘minus’ vLTSV were purified from reaction mixtures by polyacrylamide gel electrophoresis in the presence of 7 M urea. All fragments were then tested for specific self-cleavage under a wide range of conditions. The only component consistently required for self-cleavage of the RNA was a divalent metal ion, either Mg2+ or Ca2+. As an example, incubation of transcripts at 40°C in 40 m M Tris-HCl, pH 8-0, 6 mMMgCl2, for one hour showed self-cleavage of ‘plus’ ASBV (Fig. 5(C), lane 5) while no self-cleavage occurred in the absence of Mg2+ (lane 4).

In the case of purified fragment C of ‘plus’ vLTSV, self-cleavage only occurred after heating in the absence of Mg2+, snap cooling, addition of Mg2+ and incubation at 20°C (Fig. 5(D), lane 3). No self-cleavage occurred in the presence of Mg2+ without prior heating (lane 4) or on heating, snap cooling and incubation in the absence of Mg2+ (lane 5). These results were interpreted as indicating that selfcleavage could not occur in complete transcript C without prior disruption of the

Fig. 5. Self-cleavage of ‘plus’ RNA transcripts of cDNA of ASBV and vLTSV in the plasmid vector pSP64. (A) Schematic diagram of dimeric Sau3A (S) clone of ASBV constructed by excision with H indlll (FI) and EcoRI (E) from a dimeric clone in phage vector M13mp93 (Barker et al. 1985; Hutchins et al. 1986) and recloning in //w dIII and ¿icoRl cut vector pSP64. The clone was linearized with Sm al and transcribed with SP 6

RNA polymerase to give complete ‘plus’ transcript C and products 5'P, 3'P, monomer ASBV M, and fragments 5'E and 3'E. Cross-hatch, residual M13 vector; filled box, SP 6

promoter; speckles, SP 6 vector. (B) Schematic diagram of partial cDNA clone (273 nucleotides) of vLTSV in vector pSP64 (Keese et al. 1983; Hutchins et al. 1985). SP 6

RNA polymerase transcript of clone linearized with EcoRl gave complete ‘plus’ transcript C, and cleavage fragments 5 'F and 3'F. Cross hatch, vector sequences; filled box, promoter sequence. (C) Transcription products of linearized dimeric clone of ASBV analysed by autoradiography after separation on 5 % polyacrylamide 7 M urea gels. Products corresponding to A are given with positions of linear monomer ASBV marker, M, xylene cyanol F F marker dye, XC. Lane 1, transcription reaction containing only [y-3ZP]G TP as labelled nucleotide. Lane 2, labelled nucleotide [ar-32P]GTP. Lane 3, as for lane 2 but using non-linearized vector. Self-cleavage reaction products are given in lanes 4 and 5. Lane 2, purified product C. Lane 4, incubation of C in 40 mM Tris-HCl, p H 8-0, 1 mM EDTA at 0°C for 1 h (same result was obtained at 40°C). Lane 5, as for lane 4 but reaction mixture also containing 6 mMMgCl2 and incubation at 40°C for 1 h. (D) Products of transcription of linearized vLTSV clone using [ar-32P]UTP as labelled nucleotide (lane 1). Lane 2, as for lane 1 but with non-linearized vector. Purified transcript C was used for self-cleaved reactions. Lane 3, transcript C in 1 mM EDTA was heated at 80°C for 1 min, snap cooled on ice, an equal vol. 10 mM MgCl2 , 0-1 M Tris-HCl, pH 7-5, added and mixture incubated at 25 °C for 5 min. Lane 4, as for Lane 3 but without heating step. Lane 5, as for Lane 3 but without addition of Mg2+. Self-cleavage was prevented when an excess of EDTA over Mg2+ was used, or when MgCl2 was added to the RNA before the heating and snap-cooling steps. Data compiled from Hutchins et al. (1986) and Forster & Symons (1986).


native structure by heat and its partial rearrangement to a structure which allowed self-cleavage on the addition of Mg2+ (Forster & Symons, 1986). Essentially similar results were obtained with transcripts prepared from the minus partial clone of vLTSV (not given). It is important to note that the results with the partial clone of vLTSV show that only one site is required for the self-cleavage reaction.

S E L F - C L E A V A G E O C C U R S AT TWO D I F F E R E N T S I T E S I N T H E P L U S A N D

M I N U S T R A N S C R I P T S OF A S B V A N D v L T S V

The sites of cleavage of all transcripts were determined by direct enzymic sequencing (Donis-Keller et al. 1977; Haseloff & Symons, 1981). For this work, appropriate fragments were purified from non-radioactive transcription reaction mixtures or from self-cleavage reactions by fractionation on polyacrylamide gels and detection by staining. Eluted fragments could then be 5'-32P-labelled with T4 polynucleotide kinase and [y-32P]ATP at pH 9-0 without prior dephosphorylation, indicating the presence of a free 5'-hydroxyl (Silberklang et al. 1979). In the case of ASBV, enzymic sequencing showed that self-cleavage occurred between nucleotides C55 and U56, of the ‘plus’ species and between nucleotides C70 and C69 of the ‘minus’ species (Hutchins et al. 1986). For vLTSV the ‘plus’ self-cleavage site was between nucleotides A163 and U162 (Forster & Symons, 1986). (In the complementary ‘minus’ sequence, the same nucleotide numbers as the ‘plus’ sequences are retained; therefore, the ‘minus’ species are numbered in 3'- to 5'-direction).

The 3'-nucleotides at the self-cleavage sites all terminated in a 2 ',3 '-cyclic phosphate (N > p). This was shown in a number of ways. For example, ligation of [a-32P]GTP-labelled ‘plus’ and ‘minus’ products M of ASBV (Fig. 5(C), lane 2 for plus species) with a partially purified wheat germ RNA ligase preparation (Furneaux et al. 1983) gave major products which comigrated on denaturing polyacrylamide gels with the circular monomer ASBV (Hutchins et al. 1986). Wheat germ RNA ligase specifically requires a 2',3'-cyclic phosphate terminus for ligation with either a 5'-hydroxyl or a 5'-phosphorylated terminus (Furneaux et al. 1983). In addition, the production plus M of ASBV and the corresponding ‘plus’ and ‘minus’ self-cleavage fragments of vLTSV could not be labelled with [5'-32P]pCp using T4 RNA ligase, even after extensive treatment with alkaline phosphatase. However, the fragments could by labelled with 5'[32P]pCp and T4 RNA ligase after opening of the 2',3'- cyclic phosphate by incubation at 25 °C for 2h in 10 m M HC1 and treatment with alkaline phosphatase (Forster & Symons, 1986).

Enzymic sequencing of the 3 '[32P]pCp-labelled fragments showed that there was no loss or addition of nucleotides during cleavage, either on the 3'- or 5'-fragments. Hence, the reaction is a true cleavage reaction involving no loss of energy and no involvement of water since there is phosphoryl transfer from a 5'-hydroxyl to a 2 '- hydroxyl to give a 2',3'-cyclic phosphate.


+

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Fig. 6. Secondary structure models for the regions around the observed self-cleavage sites of ‘plus’ and ‘minus’ ASBV and vLTSV and ‘plus’ sTRSV and the predicted selfcleavage site of ‘plus’ vVTMoV. Arrows indicate the self-cleavage sites, and asterisks the nucleotide differences between the A and N isolates of vLTSV (Keese et al. 1983) and between vVTMoV and vSNMV (Haseloff & Symons, 1981). Boxes indicate nucleotides which are present in all structures in similar positions.In the ‘minus’ sequences, the same nucleotide residue numbers are retained as in the ‘plus’ sequences. The structures are from Keese & Symons (1987) and Forster & Symons (1987). Similar structures do not occur in ‘minus’ sTRSV and vSNMV (vVTMoV).* This is consistent with the lack of ‘minus’ monomers in VTMoV-infected plants (Fig. 1) and indicates a rolling circle model for replication as in Fig. 3(B).

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P R O P O S E D S E C O N D A R Y S T R U C T U R E S AT T H E S E L F - C L E A V A G E S IT E S OF

P L U S A N D M I N U S A SB V A N D vL TS V ARE VERY S IM IL A R

The proposed secondary structures around the self-cleavage sites of plus and minus ASBV (Hutchinses al. 1986) and vLT SV (Forster & Symons, 1986) are given in Fig. 6 together with those of the demonstrated self-cleavage site of sT SV (Prody et al. 1986) and the putative self-cleavage site ‘plus’ vVTMoV (Symons et al. 1985; Kiberstis et al. 1985). There is not only the remarkable conservation of the hammerhead structure but also the sequence homology of 17 nucleotides (boxed in Pig. 6). The structures contain three stems I, II and III, formed around an interior loop with the self-cleavage sites located in identical positions. Although stem III of ‘plus’ and ‘minus’ ASBV may appear to be thermodynamically unlikely, it may form transiently or it may be stabilized by divalent metal ions and/or by the formation of non-Watson-Crick base pairs, such as A: G base pairs, and tertiary interactions such as base triplets that occur in the three-dimensional structure of tRNA (Sanger,1984).

'The two-dimensional structures obviously do not explain why there is specific non-enzymic cleavage of plus and minus transcripts of ASBV and vLT SV . It is feasible that, in the presence of a divalent cation such as Mg2+, these structures form active tertiary complexes that lower the activation energy sufficiently and only at the internucleotide bond of the cleavage site to allow non-hydrolytic cleavage that results in phosphoryl transfer to give the characteristic 5 '-hydroxyl and 2 ',3 '-cyclic phosphate termini. The strong conservation of structure and sequence in all these


RNAs is good supporting evidence for the role of these structures and the selfcleavage reaction in the replication cycle in vivo.

O N E - T H I R D OF T H E ASBV A N D vL TSV M O L E C U L E S A R E R E Q U I R E D FO R S E L F

C L E A V A G E

In Fig. 7, the proposed secondary structures around the plus and minus selfcleavage sites of ASBV are superimposed on the proposed native structure of ASBV. Both self-cleavage structures only require local modification of the rod-like molecule of ASBV with opposite strands contributing to the formation of stems I and II (Fig. 6). It is feasible that the sole role of the central one-third of the A SBV molecule may be to provide the sequences and hence tertiary structures for the self-cleavage reactions of the ‘plus’ and ‘minus’ RNA during replication.

In the case of vL T SV , the ‘plus’ and ‘minus’ self-cleavage sites are located only six nucleotides apart at the start of the end-loop at the right-hand end of the proposed native structure (Fig. 7). Because of this, there is some overlap of the sequences required to form the ‘plus’ and ‘minus’ structures which is in contrast with the situation for ASBV. Also, considerable modification of the native structure is required to form the self-cleavage structures. However, about one-third of the molecule is needed to provide sequences for their formation and, as in the case of A SBV, this may be the only function for this part of the molecule.

IS S E L F - C L E A V A G E IN V O L V E D IN T H E R E P L I C A T I O N OF P S T V - L I K E V I R O I D S ?

On the basis of comparative sequence homology, ASBV stands apart from the remainder of the viroids that have been sequenced (Symons, 1981 ; Keese & Symons,

B vLTSV

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CUU U U U U A6 CC CAAJJ6A* UMUUCUCC u t« N IC JC UCMu«M CM SO“uCA* CUCA

CCACIMC UC 8« SUUAACUC U*CAAA«CC*« AJ*« CCM AC AMCACuC *CC CA • C •A4ocu *

Fig. 7. (A) Proposed native structure of ASBV (Symons, 1981) modified to incorporate the plus self-cleavage structure and the complement of the ‘minus’ self-cleavage of Fig. 6 (B) Location of overlapping ‘plus’ and ‘minus’ self-cleavage domains of vLTSV-A. The bars indicate that part of the molecule required to form the plus self-cleavage structure (filled) or the ‘minus’ self-cleavage structure (open). Sites of self-cleavage are indicated by arrows. From Hutchins et al. (1986) and Forster & Symons (1987).

AS BV


1986). The remainder are grouped together as the PSTV-like viroids in view of their various degrees of sequence homology with PSTV (Keese & Symons, 1987). In view of the extensive self-cleavage of in w?7ro-synthesized dimeric RNA transcripts of cDNA clones of ASB V (Hutchins et al. 1986), it was of interest to investigate similar reactions in the PSTV-like viroids. Negligible, if any, self-cleavage occurred during the in vitro transcription of monomeric or dimeric plus clones of CEV or during the incubation of purified transcripts under a variety of conditions known to induce the self-cleavage of of ASBV and vLTSV (Visvader et al. 1985). Robertson et al. (1985) have recently reported the self-processing of in vitro-synthesized dimeric RNA transcripts of cDNA clones of PSTV. However, only 1 to 5 % of the dimeric transcripts were cleaved and this occurred between nucleotides 250 and 270. In view of the low level of cleavage, it is feasible that this may have been due to random chemical hydrolysis in a sensitive region of the PSTV molecule rather than specific cleavage of the type described here.

More definitive evidence on the site of processing in the PSTV-like viroids was obtained from site-directed mutagenesis of a cDNA clone of CEV (Visvader et al.1985). Full-length cDNA clones of CEV-J (isolate J) were prepared as blunt-ended inserts (Fig. 8(A) for cloning site) in the Sm al site of the phage vector M13mp9. The cDNA clones of two of the sequence variants in this mixed isolate were restricted on either side of the insert with Sa il and£coRI, and the inserts were ligated into pSP64 restricted with the same enzymes. The DNA clones and RNA transcripts were not infectious when inoculated on to tomato seedlings. In contrast, a Bam HI cDNA plus clone of CEV-A cloned in the. Bam H l site of pSP64 (Fig. 8(A)) was infectious as was the purified insert after excision with Bam H l. Hence, the site of cloning of the CEV was important in determining infectivity. The orientation of the insert was also important since the cDNA clone containing the Bam H l insert cloned in the opposite (minus) orientation was not infectious.

Analysis of the vector sequences adjacent to the Bam H l sites of the monomeric plus CEV clone in pSP64 showed an 11 nucleotide repeat (Fig. 8(B)) of part of the viroid sequence. Hence, Bam H l clones contain a longer-than-unit-length viroid sequence. A similar 11 nucleotide repeat has been reported for infectious monomeric Bam H l cDNA clones of PSTV in the phage vector M 13mpll (Tabler & Sanger, 1984). Site-directed mutagenesis of G97 U97 and A97 was carried out in the singlestranded M13mp93 vector (Visvader et al. 1985) and the mutated insert transferred into pSP64. The intact monomeric cDNA clones plus RNA transcripts were infectious but the Bam H l excised inserts were not, in contrast to excised wild-type inserts. Most importantly, the sequence of the progeny viroid from the inoculated A97 mutant (the U97 progeny were not sequenced) was identical to wild type and not the mutant used as inoculum (Fig. 8(B)).

The most likely explanation of this result is that processing of the RNA transcripts produced in vivo occurred at one of the three phosphodiester linkages on the 3'-side of the point mutation (Fig. 8(B), positions marked by 1, 2 and 3). This would generate a full-length wild-type viroid molecule, since the point mutation lies on the 5' side of the putative 5'-processing site. The exact site of processing could possibly


be determined by site-directed mutation of the other three G nucleotides in the 11 nucleotide repeat. Reversion of the mutation to wild type is unlikely to play a role because other point mutants are non-infectious (Visvader et a l. 1985).

This work represents the first successful application of site-directed mutagenesis to study structure-function relationships in viroids.The proposed region of processing in CEV is located in a region highly conserved in the PSTV-like viroids (Haseloff et a l. 1982; Keese & Symons, 1987). There is strict conservation in these viroids of 10 of the 11 repeated nucleotides and the results imply that a function of the conserved region is the processing of longer-than-unit-length viroid RNA to unit-length viroid. However, secondary structures equivalent to those proposed for the self-cleavage of plus and minus A SBV and vL T SV (Fig. 6) have not been found in the region of the

Vector: pSP64 Clone: Infections BamHI cloning site

U/

371(372)

90IG

G A AG U C C

A100 s A A

G C C C G G G C U

UC AGG GGCCC GG A A U / a A ^ A A 270 u G

C (271) c A

Mutated CEV insert NOT infectious i-----------------------------1

CC)

170(171) 180(181)

: \ - - GCGG G A A A — G U

— CGUC CUUU — C g/A O U

m r(200)/

Blunt-end cloning site Vector: pSP64 Clone: Not infectious

Vector ■

Bam HI site l--------1 123

/ / /

Bam HI site i-------- 1 1 23

/ / /.GGAUCCCCTGGG.........................G G AU C C C C GGG G.

90 ® 89- Vector

Progeny CEV sequence from infectious cDNA clone

Fig. 8. Site-directed mutagenesis and infectivity of cDNA clones of CEV. (A) The two sites of the CEV molecule used in the construction of full-length cDNA clones. The Bam HI site was used for the cloning of CEV-A in the highly conserved central region of viroids. The blunt-end cloning site was used for the cloning of two sequence variants of CEV-J, one of which contained 372 nucleotides (residue numbers in parentheses). An A occurs at position 263 in two of the three sequence variants used while a C occurs in the other. (B) Potential in vivo processing sites of RNA transcripts derived from two point mutant clones of CEV-A. The circle nucleotides represent the point mutations introduced into CEV-A at position 97, resulting in CEV-A/U97 and CEV-A/A97. The B am \l\ cloning sites and adjacent sequences are shown. The 11 nucleotide repeat sequence that correlates with infectivity and the proposed sites of processing at positions 1, 2, or 3 are indicated, as well as data on infectivity on tomato seedlings. Figures modified from Visvader et al. (1985).


putative processing site of CEV and the other PSTV-like viroids. Hence, it is feasible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle.

The work from this laboratory described here was supported by the Australian Research Grants Scheme and by Commonwealth Government Grant to the Adelaide University Centre for Gene Technology in the Department of Biochemistry. We thank Jennifer Cassady and Tammy Edmonds for assistance and Leanne Goodwin for typing the manuscript.

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self-cleavage of rna in the replication of viroids …self-cleavage of viroid and virusoid rna 307 a...

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