Formation of Circular Satellite Tobacco Ringspot Virus RNA in Protoplasts Transiently Expressing the Linear RNA

VIROLOGY

239, 413±425 (1997) VY978897

ARTICLE NO.

Formation of Circular Satellite Tobacco Ringspot Virus RNA in Protoplasts Transiently Expressing the Linear RNA Catherine A. Chay, Xia Guan, and George Bruening1 Department of Plant Pathology, College of Agricultural and Environmental Sciences, University of California, Davis, California, 95616 Received September 2, 1997; returned to author for revision September 19, 1997; accepted October 9, 1997 The most abundant form of the satellite RNA of tobacco ringspot virus (sTRSV RNA) is a linear, unit length molecule of 359 nucleotide residues, designated L2(1)M. A postulated replication scheme for the satellite RNA has as its first, and apparently virus-independent, step the ligation of L2(1)M into the corresponding circular form C2(1)M. We transiently expressed L2(1)M wild type and L2(1)M mutants in tobacco protoplasts using an African cassava mosaic geminivirus vector. Measured extents of C2(1)M accumulation were correlated with computer-predicted folding to suggest wild-type secondary structure elements that might be deleted without reducing ligation. A 127-nucleotide residue mutant L2(1)M was created by replacing, with 7 and 3 residues, respectively, nucleotide residues 53±211 and 268±350, each of which was predicted to form a set of three adjacent imperfect stem-loops in wild-type L2(1)M. The mutant L2(1)M was found to be extensively ligated to C2(1)M in protoplasts and to retain a calculated helix of the wild-type molecule that incorporates the 39 terminal sequence. A trinucleotide in the 39 region was mutated so as to disrupt and restore, respectively, the calculated helix, reducing and restoring, respectively, C2(1)M formation. These results suggest that the 39 stem contributes to the suitability of the small L2(1)M molecules as a substrate for a protoplast RNA ligase and that computed folding of sTRSV RNA may be predictive of sTRSV RNA structure in vivo. © 1997 Academic Press

C2(2)M. However, only linear RNA, including multiple copies of L2(1)M TRSV (Schneider et al., 1972) and oligomeric sTRSV RNAs, were detected in RNA from virion preparations (Passmore and Bruening, 1993). When incubated in vitro in a suitable buffer, dimeric and trimeric sTRSV(1)RNAs from virion preparations and in vitro transcripts of both polarities self-cleave at a specific phosphodiester junction. Cleavage occurs between nucleotide residues 359 and 1 of succeeding L2(1)M units and between residues 49 and 48 in oligomeric sTRSV(2)RNA, both sequences numbered according to sTRSV(1)RNA (Buzayan et al., 1986b, 1986a; Prody et al., 1986). The subsets of sTRSV RNA nucleotide sequences that mediate cleavage reactions have been identified, and the ends newly created by cleavage are a 59-hydroxyl and a 29,39-cyclophosphate (Buzayan et al., 1988; Feldstein et al., 1989; Forster and Symons, 1987; Haseloff and Gerlach, 1988, 1989). L2(1)M and L2(2)M recovered from extracts of infected tissue appear to have the same structure as the corresponding RNAs derived by self-cleavage of in vitro transcripts, suggesting that the self-cleavage reactions observed in vitro also occur in vivo (Passmore and Bruening, 1993). The sTRSV(2)RNA sequence exhibits not only self-cleavage but also a spontaneous ligation reaction in vitro, and purified L2(2)M or purified C2(2)M spontaneously equilibrate to a mixture of L2(2)M and C2(2)M (Buzayan et al., 1986b; Feldstein and Bruening, 1993; Passmore and Bruening, 1993). In contrast, L2(1)M and

INTRODUCTION The first virus satellite RNA to be investigated was the satellite RNA of tobacco ringspot virus (sTRSV RNA) (Schneider, 1969, 1971). sTRSV RNA has been found in association with several TRSV isolates and generally reduces the titer of the associated TRSV and the severity of TRSV-induced symptoms, whether sTRSV RNA is inoculated or introduced transgenically (Gerlach et al., 1987; Passmore et al., 1995; Schneider, 1977). The presence of both polarities of the satellite RNA was demonstrated by the purification of the corresponding doublestranded RNA from tissues infected by TRSV and sTRSV RNA (Schneider, 1977). Messenger activity has not been associated with either polarity of sTRSV RNA (Owens and Schneider, 1977; Ponz et al., 1987), and convention designates the most prevalent polarity sTRSV RNA as the (1) polarity. The most abundant encapsidated sTRSV RNA species is the 359 nucleotide residue, linear, unit length, ``monomeric'' L2(1)M (Fig. 1A). Infected tissue also accumulates complementary polarity L2(2)RNA, repetitive sequence dimers and higher oligomers of both polarities (Kiefer et al., 1982), and circles of both polarities (Bruening et al., 1991; Linthorst and Kaper, 1984; Sogo and Schneider, 1982), designated C2(1)M and C2(2)M. L2(1)M and C2(1)M are about equally abundant in extracts of infected tissue, as are L2(2)M and

1

To whom reprint requests should be addressed. 413

0042-6822/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.

414

CHAY, GUAN, AND BRUENING

FIG. 1. Computed secondary structures of wild-type and mutant sTRSV RNA sequences. (A) Secondary structure of the reference, unpermuted sTRSV RNA strain T sequence (Passmore et al., 1995)). Nucleotide sequence numbering is according to Buzayan et al., (1986a). (B) Computed secondary structure of the hammerhead self-cleaving sequence of sTRSV RNA. J(1) is the scissile phosphodiester junction, between nucleotide residues 359 and 1. (C) Partial representation of computed secondary structures of the mutant sTRSV RNA sequences D-16, D-17, etc., showing only the stem or stems of each structure that incorporate all or part of nucleotide residues 350±359. Asterisks indicate mutants that were tested in plants but not protoplasts. Regions of the hammerhead structure [part B; (Hertel et al., 1992)] are marked by boxes for helix-loop I (negative contrast), helix-loop II (shaded), and helix III (unshaded), which are keyed to the corresponding sequences of sTRSV RNA and its mutants in parts A and C. Folded regions of the computed sTRSV RNA structure are defined here by sequence ranges in parentheses: I (1±19), A (20±53; 211±256), B (54±70), C (71±175), D (176±210), E (271±295), F (298±320), G (324±348), and uninterrupted helix H (261±270; 350±359).

C2(1)M equilibrate to only a very limited extent (Passmore and Bruening, 1993; Prody et al., 1986). The selfcleavage and spontaneous ligation reactions of sTRSV RNA almost certainly have a role in replication (van Tol et al., 1991; Sheldon and Symons, 1993). The documented RNA species and reactions of sTRSV RNA suggest synthesis occurs by rolling circle transcription (Bruening et al., 1988; Kiefer et al., 1982; Symons et al., 1985) and favor a symmetrical model in which the likely first step is enzymatic ligation of L2(1)M to C2(1)M (Bruening, 1990; Bruening et al., 1991; Symons, 1992). This reaction is not dependent on TRSV, as indicated by generation of C2(1)M in plants transgenically expressing L2(1)M (Robaglia et al., 1993). Rolling circle transcription, almost certainly dependent on activities present only in TRSV-infected tissue, generates selfcleaving oligomeric sTRSV(2)RNA, generating L2(2)M. Spontaneous ligation of L2(2)M is presumed to begin the second half of the replication cycle, which, when completed by rolling circle transcription and self-cleavage, generates new copies of L2(1)M.

In work reported here, L2(1)M was generated in protoplasts and plants by transient expression from a geminivirus vector in which most of the coat protein open reading frame (ORF) was replaced by sTRSV RNA sequences (Fig. 2A). This system was used to express wild-type and several mutant versions of L2(1)RNA, without a requirement for inoculation of TRSV. C2(1)M formation was retained for mutants designed so as to remove sequences composing specific calculated elements of secondary structure, suggesting that at least some aspects of the predicted folding have relevance in vivo. MATERIALS AND METHODS Constructions for expression of subdimeric sTRSV RNA sequences Full-length clones of ACMV (West Kenyan isolate) genomic components in bacteriophage M13 vectors were obtained from Dr. John Stanley (Stanley, 1983): DNA B, pET094; DNA A, pET092 and pET012, the latter having

SATELLITE RNA CIRCLES

an EcoRV site replacing a 727-bp DNA fragment of the coat protein ORF [(Ward et al., 1988); nucleotide residues 467±1194]. Numbering for ACMV sequences is as designated (Stanley and Gay, 1983). Molecular cloning procedures were as described (Sambrook et al., 1989). Paul A. Feldstein of this laboratory transferred full-length ACMV sequences to plasmid vectors from the indicated bacteriophage M13 clone cut at the ACMV restriction sited identified in parenthesis: to pIBI, from pET092 (MluI, 734), generating pETA092; to pTZ18R (U.S. Biochemicals), from pET094 (PstI, 245), generating pETB094; to pTZ18R, from pET012 (BamHI, 291), generating pETA012. Plasmid pTTS-B (Haseloff and Gerlach, 1989) was derived from vector pGEM4 and a circularly permuted, subdimeric, 522-bp sTRSV RNA sequence flanked at its 59 end by a bacteriophage T7 promoter and restriction endonuclease sites HindIII, SphI, and PstI and flanked at its 39 end by restriction endonuclease sites SstI and EcoRI and a bacteriophage SP6 promoter. The subdimeric sTRSV RNA sequence extends from the unique TaqI site at nucleotide residue 277 through one circularly permuted 359-bp unit sequence to a second TaqI site and then to the unique SpeI site at nucleotide residue 81. Thus, the duplicated portion of the sTRSV RNA sequence encompasses two hammerhead (Forster and Symons, 1987) self-cleaving sequences. The self-cleaving sequences released an unpermuted monomeric sTRSV RNA molecule from in vitro transcripts (not shown). The sTRSV cDNA insert of pAC1.0TTSB was prepared by polymerase chain reaction (PCR) amplification of a 692-bp DNA fragment from plasmid pTTS-B. Primers dAATTCGAATTAATACGACTCACTATAG (designated T7-8, encompassing a bacteriophage T7 promoter) and dCTAGAGGCTTGTACATATTGTCGTTA (designated Xba-GEM4) were 59-phosphorylated. PCR conditions were 10 mg/ml plasmid pTTS-B, 1 mg/ml each primer, 200 mM each dNTP, 50 mM KCl, 10 mM Tris±HCl, pH 8.3, 1.5 mM MgCl2, 10 U/ml Taq DNA polymerase (Perkin±Elmer), 25 cycles of 1 min at 94°C, 1 min at 45°C, and 2 min at 72°C. Electrophoretically purified PCR product was ligated into EcoRV-digested and phosphatase-treated plasmid pETA012/Bam. Plasmid pETA012/Bam is identical to pETA012 except that all restriction endonuclease sites of the pTZ18R polylinker, except BamHI, were removed. Clones were screened by colony PCR using T7-8 and Xba-GEM4 primers to identify pAC1.0TTSB. pAC1.3TTSC (Fig. 2A) has 1.3 copies of ACMV A DNA, with two origins of replication and subdimeric sTRSV cDNA replacing most of the ACMV coat protein ORF. It was constructed from pAC1.0TTSB and pAC0.3C. pAC0.3C was prepared by inserting a vector bearing a chloramphenicol resistance gene at the ClaI site of ACMV DNA A (coordinate 2403), digesting the resulting plasmid clone to completion with BamHI (coordinate 291) and partially with NcoI (2124), and recovering the DNA fragment that had the vector inserted into the ACMV DNA

415

A region 2124±2779/1±291. This fragment was bluntended and ligated to create pAC0.3C. pAC0.3C was digested with BamHI, phosphatase treated, and ligated into BamHI-digested pAC1.0TTSB. Clones with a headto-tail orientation within the 1.3 units of A DNA sequence were selected as pAC1.3TTSC. To create plasmid pAC1.0SSS, a dimeric cDNA of sTRSV RNA (Fig. 1A), circularly permuted about the Sau3A site (nucleotide residue 245), was excised by digestion of p651-4 (Ponz et al., 1987) with restriction endonucleases BamHI and EcoRI. The released 732-bp DNA fragment, which included flanking vector sequences, was made blunt ended and was cloned into EcoRV-digested pETA012. Plasmids screened for orientation of insert expected to generate positive polarity sTRSV RNA from the ACMV coat protein promoter. Replacement mutations of the monomeric sTRSV RNA sequence Dr. J. Haseloff provided a set of D-series of mutants, which except for D-17 have a region of the central, unpermuted, unit length sTRSV RNA sequence of pTTS-B replaced by a BamHI linker sequence, CGGATCCG (Table 1). D-17 has an insert of 16 residues composed of a BamHI linker sequence and a repeat of the 72±79 sequence of sTRSV RNA [(Haseloff and Gerlach, 1989); Table 1]. D-17/43, D-22/25, D-22/47, D-25/22, D-47/22, and D-99/16, all of which have the indicated BamHI linker sequence, were constructed by combining the sequences of plasmid pairs at the BamHI site. Nucleotide sequence numbering for wild-type sTRSV RNA (Fig. 1A) was retained for the mutants. Plasmid with cDNA insert encoding mutant XF1 (Table 1) was created from pAC1.3TTSC as template using an overlap extension polymerase chain reaction method (Ho et al., 1989) to alter the central, unit length sTRSV RNA. Primers for one reaction were T7-8 and dGAAGTCGACACCCGACAGTCCTGTTT and for the other were dGTCGGGTGTCGACTTCTCTGTTTGT and Xba-GEM4. PCR conditions were as above but with 4 cycles of 1 min at 94°C, 1 min at 42°C, and 1 min at 72°C and 20 cycles with temperatures 94, 55, and 72°C. The two DNA fragments were electrophoretically purified, combined, and subjected to PCR amplification using the T7-8 and Xba-GEM4 primers. Electrophoretically purified DNA product was digested with HindIII and SstI and was ligated to similarly digested but also phosphatase-treated plasmid pTZ19R. XF1 has sTRSV RNA nucleotide residues 53 through 209 replaced by GGGUGUC, which with the GAC of nucleotide residues 210±212 creates a SalI site. In XF2 (Table 1) nucleotide residues 274±348 of XF1 are replaced by A, creating an XbaI site. In XF3 nucleotide residues 268±350 of XF1 are replaced by CGG, creating a KpnI site. XF2 and XF3 were prepared similarly. For construction of XF3 (Ho et al., 1989), the tem-

416

CHAY, GUAN, AND BRUENING

plate was the XF1 clone in pTZ19R and primers were (dAACAGCTATGACCATG) (reverse) with dGACAGGGTACCGCCCCGTCGCCGCTAATG and dGACGGGGCGGTACCCTGTCACCGGATG [oXF3(14)] with dGTAAACGACGGCCAGT (universal). In mutants XG4 and XG5, the XF3 sequence ACG at coordinates 262±264 was replaced by UGC and GCA, respectively. For construction of XG4, two PCR products were prepared from XF3 template. The product from a reaction with dGCGGGTACCGCCCGCACGCCGCTAATGCGAGATC and reverse as primers was electrophoretically purified and digested with KpnI and HindIII. The purified product from oXF3(14) and universal primers was digested with KpnI and EcoRI. The two digested PCR products and HindIIIand EcoRI-digested pTZ19R were ligated to obtain a clone of XG4. XG5 was constructed similarly except that the first primer was dGCGGGTACCGCCCTGCCGCCGCTAATGCGAGATC. Versions of pAC1.3TTSC with mutations of the nonpermuted, unit sTRSV RNA sequence were prepared by digesting the plasmid with HindIII and SstI and treating with phosphatase. The insert of mutant sTRSV cDNA was derived from the appropriate D-series pGEM4 clones or X-series pTZ19R clones or from the PCR-amplified products thereof. Cloning was into HindIII and SstI sites, with selection on chloramphenicol-containing medium. The pAC1.3TTSC-derived clones of the sTRSV RNA mutant set XF3, XG4, and XG5 were modified by insertion, as a fusion to the 59 side of the subdimeric sTRSV RNA sequence, of a Nicotiana rustica tRNATyr sequence. This introduced a RNA polymerase III promoter known to be active in protoplasts (Perriman et al., 1995; Stange and Beier, 1986). pGtRNA (from Dr. Rhonda Perriman) was cut with SstI and PstI to release the insert, which was cloned into similarly cut and then phosphatasetreated pSportI (BRL). The 59 portion of the tRNA gene was amplified by PCR using universal primer and the HindIII cleavage site-containing primer dCACTAGTCTGCAGCAAGCTTCTACCGGATTCGAACCAG. The 39-most 18-nucleotide residues of the last primer are complementary to a region of the tRNA gene immediately to the 59 side of the RNA polymerase III termination site. The PCR product was digested with HindIII, and the fragment was ligated to HindIII-cut XF3, XG4, and XG5 clones derived from pAC1.3TTSC. Clones with the proper orientation of insert were selected by restriction mapping. Preparation, culture, and inoculation of protoplasts and plants Culture of Nicotiana tabacum cv Xanthi nc suspension cells, in a culture medium based on Murashige and Skoog minimal organic medium (Gibco-BRL 510-1118EB), and preparation of protoplasts both were as reported (Passmore and Bruening, 1993). Protoplasts were sus-

pended in electroporation solution (200 mM mannitol, 10 mM HEPES, pH 7.2, 10 mM sodium chloride, 4 mM calcium chloride, 120 mM potassium chloride) to a density of 1 3 106/ml. Seven hundred microliters of protoplast suspension were mixed in a Hoeffer DispoProGenitor 4-mm cuvette with 100 ml of electroporation solution containing 5 mg of supercoiled plasmid preparation (pAC1.3TTSC or its derivative, equivalent to about 2 mg of ACMV DNA A insert). Electroporation was in a Hoeffer Progenetor II with a current pulse under settings of 330 V, 500 mFarad, 8 ms in experiments with D-series mutants and 270 V, 760 mFarad, 8 ms in experiments with X-series mutants. Samples were placed on ice for 10 min following electroporation, transferred to petri dishes with 9.2 ml of filter sterilized growth medium [a 19:1 mixture of 265 mM mannitol in culture medium and coconut water (Gibco), pH 5.8, 0.5 mg/ml cephaloridine]. Petri dishes were sealed with parafilm and incubated in the dark without shaking. ACMV-derived plasmids also were inoculated to Nicotiana benthamiana plants. Plasmid pAC1.3TTSC, or its derivative with mutated sTRSV RNA sequence, was coinoculated with the wildtype DNA B clone, pETB094, that had been digested with PstI to release the DNA B insert. Plasmid corresponding to the equivalent of 1 mg of ACMV DNA was dissolved in 10 ml of sterile water and was rubbed on the two youngest leaves of three week old, 5-7 leaf stage N. benthamiana plants that had been dusted with diatomaceous earth. Plants were maintained in growth chambers at 25°. Extraction and analysis of protoplast and plant nucleic acids Cultured protoplasts were harvested at times indicated by centrifugation at 600 g for 6 min, and the supernatant was discarded. RNA was extracted either directly by application of the Tri (Molecular Research Center, Inc.) or Trizol (Gibco-BRL) reagent (Chomczynski, 1993) as described (Passmore et al., 1995), or collected protoplast precipitates were frozen at 280°C. Frozen samples were thawed (approximately 200 ml) and diluted to 1 ml with TE buffer (10 mM Tris±HCl, 1 mM EDTA, pH 8) containing 100 mM NaCl and 5 mg/ml SDS. Samples were extracted three times with an equal volume of (1:1) phenol:chloroform, and nucleic acids were precipitated by additions of sodium acetate and ethanol. For RNA analysis, total nucleic acid samples were treated with RQ1 DNAse for 1 hr at 37°C in 40 mM Tris±HCl, pH 7.9, 10 mM NaCl, 6 mM MgCl2 and were extracted with an equal volume of (1:1) phenol:chloroform and precipitated. DNA samples from plants, or total nucleic acid samples from protoplasts, were fractionated on 1.4% agarose gels in 1X TBE (Sambrook et al., 1989). Gels were soaked in 0.25 M HCl for 10 min and capillary blotted to Hybond N1 membranes (Amersham) in 0.4 M NaOH. Mem-

SATELLITE RNA CIRCLES

branes were rinsed in 2X SSC (1X 5 0.15 M NaCl, 15 mM sodium citrate, pH 7), 1 mg/ml SDS and air dried. RNA samples were dissolved in water or TE and were diluted into an equal volume of formamide, heated to 80°C for 2±3 min, and subjected to electrophoresis through 6.5% polyacrylamide (24:1, acrylamide:bis-acrylamide) gel in 7 M urea at 5 watts in a Bio-Rad MiniProtean II apparatus. Transfer to Hybond N1 membrane was as described (Passmore and Bruening, 1993) except that electroblotting (Bio-Rad Mini Trans-Blot) was at 4°C and 20 V overnight. RNA probes of (1) polarity and (2) polarity were prepared as described (Davanloo et al., 1984; Passmore and Bruening, 1993) from plasmid p20SD(2)3 (Ponz et al., 1987). DNA probe was generated using a multi-prime labeling reaction kit (Amersham) and BamHI-digestedpAC1.3TTSC or electrophoretically purified ACMV DNA A fragment from MluI-digested plasmid pETA092. Blot hybridization was as described (Church and Gilbert, 1984), except that the BSA was omitted and deionized formamide was added to a final concentration of 50% (Passmore and Bruening, 1993). Blots were prehybridized for at least 1 h at 55°C and hybridization was carried out overnight at 55°C for RNA probes and at 42°C for DNA probes, each provided at 106 cpm/ml. Blots were washed under stringent conditions, and RNA blots were treated with 1 mg/ml RNAase A at 37°C for 1 h. Background-corrected radioactivity of bands was calculated from results of two-dimensional b-particle counting with an AMBIS Radioanalytic Detector (Automated Microbiological Systems, Inc., San Diego, CA). The radioactivities associated with C2(1)M and L2(1)M bands were used to calculate the ratio C2(1)M/[C2(1)M 1 L2(1)M] for both mutant and wild-type satellite RNAs, and results were normalized by dividing the former ratio by the latter ratio. The FOLD program (Genetics Computer Group, Inc., Madison, WI) was used to predict secondary structures of sTRSV RNA and its mutants (Devereux et al., Smithies, 1984). C2(1)M was prepared in vitro as described (Buzayan et al., 1995) from L2(1)M that had been electrophoretically purified from transcript of plasmid pT7(1)117/M/D synthesized in a bacteriophage T7 RNA polymerase-catalyzed reaction. RESULTS Expression of wild-type sTRSV RNA from ACMVderived vector Plasmids for expression of sTRSV RNA in protoplasts were constructed from an ACMV-based vector, pAC1.3TTSC (Fig. 2A). pAC1.3TTSC has two copies of the ACMV A DNA common region, and most of the ACMV coat protein open reading frame is replaced by a subdimeric sTRSV RNA sequence. Suspensions of approximately 125,000 protoplasts were separately electroporated with 2 mg of uncut pAC1.3TTSC, BamHIdigested pAC1.0SSS, or MluI-digested pETA092. pAC1.0SSS and pETA092 have only one common region, the latter also

417

differing from pAC1.3TTSC because it lacks sTRSV RNA-derived sequence. At 3±4 days after electroporation, each construction engendered a similar amount of supercoiled, ACMVderived DNA (data not shown). Thus, for protoplasts inoculated under these conditions, the uncut, two-commonregion plasmid pAC1.3TTSC and released unit length ACMV derivative from pETA092 or pAC1.0SSS were similarly effective as inocula. The insertion of cDNA for transcription of sTRSV(1)RNA did not prove to be a significant burden on accumulation of ACMV-derived DNA in protoplasts. pAC1.3TTSC DNA was detected in protoplast extracts immediately after electroporation, but at 3 days postelectroporation the principal band detected by hybridization corresponded in its mobility to supercoiled ACMV A DNA and in amount to roughly half the protoplast-associated inoculum at electroporation (Fig. 2D, lanes 1 and 2, with correction for amounts of protoplasts). The ACMV A DNA continued to accumulate after the third day (Fig. 2D, lane 3). The two self-cleaving sequences (Figs. 1B and 2A) of the expected circularly permuted, subdimeric sTRSV RNA transcript of the pAC1.3TTSC should release L2(1)M by self-cleavage of the in vivo transcript (Fig. 2B). The new ends, a 29,39-cyclophosphate and a 59-hydroxyl group, are appropriate for L2(1)M to serve as substrate for a RNA ligase of a type found in wheat germ extract (Konarska et al., 1982; Schwartz et al., 1983). As expected (Figs. 2B and 2C), sTRSV RNA of both the linear, monomeric form, L2(1)M, and the corresponding circular form, C2(1)M, appeared in extracts of the inoculated protoplasts (Fig. 2E). Depending on the condition of the protoplasts, sTRSV RNA either declined at later times after electroporation (Fig. 2E, lane 3) or continued to accumulate (not shown). The flanking 240- and 321-nt fragments (Fig. 2B) generally were not detected in the extracts of electroporated protoplasts (Fig. 2E), presumably due to instability of these incomplete sTRSV(1)RNA molecules. Coinoculation of protoplasts with TRSV and plasmid pAC1.3TTSC resulted (not shown) in infections that accumulated encapsidated sTRSV RNA. N. benthamiana plants were coinoculated with PstI-cut pETB094 as a source of ACMV DNA B and with either BamHI-digested pAC1.0SSS or MluI-digested pETA092. Three weeks later, the plants were inoculated with TRSV. Systemically infected tissue was collected after 1 week. Virion preparations from the pAC1.0SSS-inoculated, but not the pETA092-inoculated, plants included encapsidated sTRSV RNA (not shown). Thus, transiently expressed wild-type sTRSV(1)RNA was supported for replication by TRSV. The observed biological activity speaks to the authenticity of the central, unpermuted, monomeric sTRSV RNA sequence of pAC1.3TTSC and the other ACMV-derived plasmids.

418

CHAY, GUAN, AND BRUENING

FIG. 2. Expression of sTRSV RNA in tobacco protoplasts after inoculation of a permuted, subdimeric copy of sTRSV RNA cDNA as an insert in a vector derived from African cassava mosaic geminivirus (ACMV). (A) Structure of plasmid pAC1.3TTSC, which incorporates into the plasmid vector p129C approximately 1.3 unit lengths of the ACMV A DNA sequence, such that two copies of the common region (COM) are included. Most of the ACMV coat protein open reading frame (ORF) was replaced by a permuted subdimeric copy of the sTRSV RNA sequence [shaded rectangles with self-cleavage sites J(1)] flanked by bacteriophage T7 and SP6 promoters. The remaining portions of the coat protein ORF are represented by two black rectangles. (B) Diagram of the transcript and self-cleavage products expected to accumulate in protoplasts transfected with pAC1.3TTSC: linear, unit length sTRSV RNA, L2(1)M, and two flanking RNA molecules of 321 and 240 nt. (C) Ligation of L2(1)M is expected to yield the circular, unit length sTRSV RNA C2(1)M. (D) Analysis of ACMV DNA accumulating after electrophoration of protoplasts with pAC1.3TTSC. Nucleic acids were purified from protoplasts directly after electroporation (lane 1, corresponding to 20,000 protoplasts) and at 3 and 5 days after electroporation (lanes 2 and 3, respectively, corresponding to 40,000 protoplasts each). Electrophoresis was through 1.4% agarose gel. Detection was by blot hybridization with cloned ACMV DNA probe and autoradiography. The arrow locates the expected position of supercoiled ACMV DNA. (E) Analysis of sTRSV RNA accumulating in protoplasts electroporated with plasmid pAC1.3TTSC. Conditions are as for part D except that nucleic acids were subject to electrophoresis through 6.5% polyacrylamide gel, electroblotting, and hybridization to transcribed single-stranded RNA probe for sTRSV(1)RNA. Migration positions of sTRSV RNAs C2(1)M and L2(1)M are indicated and were located using standards derived by ligation of L2(1)M in wheat germ extract.

Correlation of C2(1)M accumulation with computed elements of secondary structure A set of the 14 sTRSV RNA replacement mutants, designated as the D-series (Fig. 3A), was selected to provide, in aggregate, alterations over most of the central, unpermuted L2(1)M sequence (Fig. 2B), exclusive of the sequences that mediate self-cleavage (Fig. 1B). The mutants were either transferred from existing clones (Haseloff and Gerlach, 1989) or were derived by combining pairs of such mutant clones. Most of the mutants, when inoculated to protoplasts as their pAC1.3TTSC derivative, engendered significant accumulation not only of the mutant L2(1)M but also of the corresponding C2(1)M (Fig. 4). In each experiment, we inoculated pAC1.3TTSC bearing wild-type as well as the mutant sTRSV RNA sequences so as to provide a reference for the extent of accumulation of wild-type C2(1)M in that experiment. The extent of C2(1)M formation was calculated as the fraction of accumulated C2(1)M relative to accumulated L2(1)M and C2(1)M. When results, from 3 to 7 experiments for each mutant, were expressed as the ratio of mutant fraction divided by wild-type fraction, the variation in the accumulation ratio from experiment to experiment was small enough to allow separation of the results for different mutants into statistically distinct groups (Fig. 3B).

For half of the mutants tested in protoplasts, the accumulation ratio for C2(1)M was close to 1.0 [Fig. 3B, group a, and Fig. 4]. Testing of some mutants was performed in N. benthanmiana plants, by coinoculation of the pAC1.3TTSC plasmid with PstI-cut plasmid pETB094 as the source of ACMV DNA B (mutants identified by asterisks, Fig. 1C; deletions diagrammed in Figs. 5A and 3A). For the three D-series mutants examined in both protoplast and plant systems, D-75, D-99/16, and D-22, the results were similar, with only D-22 failing to give C2(1)M in good yield relative to the wild-type control (Figs. 3B, 4, and 5B). Five other D-series mutants tested only in N. benthanmiana showed accumulation of C2(1)M comparable to or greater than what was observed for the wild-type control (Fig. 5B and data not shown). Reduced accumulation of C2(1)M relative to L2(1)M was associated with more than one region of the sTRSV RNA sequence (Figs. 3 and 5). Therefore, we also examined computed secondary structures for the mutant and wild-type sequences and compared them with the corresponding extents of C2(1)M accumulation in vivo. As expected, each calculated secondary structure (not shown) consisted of portions that could be superimposed on the structure for wild-type sTRSV RNA and other portions that were altered. Figure 1A defines a set

SATELLITE RNA CIRCLES

419

FIG. 3. Deletion and substitution mutants of sTRSV RNA and their extents of ligation to circles after expression from ACMV vector in protoplasts. (A) Diagram of unit length sTRSV RNA L2(1)M with mutations, represented by thin lines, and inserted BamHI linker-derived sequence (CGGAUCCG), represented by shaded box. Unshaded boxes identify the sequences retained in each construction for mediating self-cleavage (Fig. 1B) and release of the unit length RNA molecules. (B) Accumulation of circles in protoplasts in which mutant sTRSV RNA sequences were expressed. Analyses were as presented in Fig. 4. The radioactivity ratio C2(1)M/[C2(1)M 1 L2(1)M] was calculated for mutant and wild-type sTRSV RNA in each experiment. The mutant ratio divided by the wild-type ratio was averaged, within each experiment, from results from at least three independent electroporations, 1/2 the standard deviation. Letters in parentheses and shading of the bar graph define groups whose members are not statistically distinct.

of secondary structure regions, I, A, B, ... H, of a computer-derived secondary structure of wild-type sTRSV RNA. The regions that were altered in the computed structures of each of the D-series mutants are indicated in Table 1. For example, the computer-derived secondary structure of D-99/16, the most extensively deleted mutant of Fig. 3A, retained the folding of the wild-type sequence for residues 1±19 (stem loop I of Fig. 1A) and 257±359

FIG. 4. Analysis of accumulated sTRSV RNA C2(1)M and L2(1)M in protoplasts. Protoplasts were electroporated with ACMV A DNAderived plasmids having a cDNA insert derived from the D series sTRSV RNA deletion and substitution mutants (as indicated above lanes and defined in Table 1 and Fig. 3) or intact sTRSV RNA (pAC1.3TTSC, designated T). RNA was extracted from protoplasts 4 days after electroporation, and approximately 10 mg were applied to each lane for analysis as in Fig. 2E. Ranges of migration positions for C2(1)M and L2(1)M are indicated.

(structures E, F, G, and H of Fig. 1A). Elsewhere (structures A, B, C, and D), the calculated folding of the two molecules was distinct. Reduced C2(1)M formation from L2(1)M was associated with an altered stem-loop I for three mutants, D-60, D-47, and D-22. Eleven D-series mutants (D-17, D-99, D-99/16, D-16, D-17/43, D-94, D-88, D-25, D-25/22, D-9, and D-22/47) had an intact stem-loop I and showed C2(1)M formation to an extent comparable to that of wild-type sTRSV RNA. Thus, for the 19 mutants tested, 14 showed either (i) reduced C2(1)M formation and an altered stem-loop I or (ii) an extent of C2(1)M formation comparable to the control and unaltered stem-loop I. By difference, 5 of 19 results were exceptions to this correlation. Table 1, entry line 20, summarizes the exceptions to this correlation for the secondary structure region set I, A, B, ... H. The fewest such exceptions, 2, were found for stem H (Table 1): mutants D-101 and D-22/47. Nine of the 19 D-series mutants tested in protoplasts or plants retained structure region H, a 10-bp stem that incorporates residues 261±270 and 350±359. This 39-most helix is the longest uninterrupted stem of the calculated sTRSV RNA structure (Fig. 1C, first group of listed mutants). Additionally, the D-9 structure retained a version of stem H that is truncated by one base pair, and the D-25 and D-25/22 structures had the H helix extended to 11

420

CHAY, GUAN, AND BRUENING

FIG. 5. Analysis of accumulation of sTRSV RNA C2(1)M and L2(1)M in N. benthamiana plants coinoculated with double-stranded ACMV B DNA (PstI-digested pETB094) and double-stranded ACMV A DNA (wild-type and sTRSV RNA-mutant versions of pAC1.3TTSC). (A) Diagrams defining the deletion and substitution mutations in the unpermuted, unit length sTRSV RNA sequence, using the conventions of Fig. 3A. (B) Analysis of accumulation of linear and circular sTRSV RNA in extracts of N. benthamiana tissue, under conditions described in the legend to Fig. 4. Figure 3A defines mutants D-22, D-75, and D-99/16, which are not defined in part A.

bp. The calculations for these 12 mutants all showed extensive variations from the wild-type sTRSV RNA folding pattern elsewhere in the sequence, with nine different sets of secondary structure regions affected (Table 1). Of the 12 mutants with intact, truncated, or extended stem H, all except D-101 (Fig. 3B) showed accumulation of C2(1)M comparable to the wild-type accumulation. Effects of deletions corresponding to groups of predicted secondary structure elements The analysis described above suggested that construction XF1, with computed secondary structure regions B, C, and D deleted (Table 1 and Figs. 1A, 6A, and 7A), readily should generate C2(1)M from transiently expressed L2(1)M. XF1 C2(1)M accumulation in protoplasts was at least as efficient as wild-type C2(1)M accumulation (Fig. 6C, lanes 1 and 2). XF2 was constructed from XF1 with the additional deletions of secondary structure regions E, F, and G (Table 1). The selfcleavage of the subdimeric precursor of XF2, incubated in vitro, was limited, so XF2 was not tested by transient expression in protoplasts. We suspected that replacing the E, F, and G secondary structure regions of XF1 with a small loop stabilized the full 10-base-pair stem H (Fig. 1A) so effectively that it competed well with the structure necessary for the self-cleavage reaction (Fig. 1B). There-

fore, XF3 was constructed, for which the predicted secondary structure has a truncated 7-base-pair version of stem H (Table 1 and Fig. 7B). In initial attempts (not shown) to express XF3 transiently from ACMV vector in protoplasts, accumulation of XF3 was very limited, as detected by blot hybridization. Reliable signals for L2(1)M and C2(1)M were not obtained after blot hybridization analyses of protoplast extracts. Previous experiments had shown a good correlation between the extents of conversion of L2(1)M to C2(1)M for various mutants of the D-series when they were expressed transiently in protoplasts and when in vitro-transcribed RNA was incubated with wheat germ extract and rATP (not shown). Therefore, XF1, XF2, and XF3, as well as the substitution mutants of XF3, namely XG4 and XG5, designed to alter the stability of stem H, were incubated in wheat germ extract (Figs. 6A, 6B, and 7; XF2 result not shown). XG4 was partially degraded during the incubation. Several ribonuclease inhibitors at several concentrations were included in reaction mixtures in order to identify conditions under which XG4 would be protected but the ligation reaction would proceed (not shown). The most suitable condition was achieved by addition of vanadyl nucleosides to 0.3 mM. Lesser concentrations of vanadyl nucleosides were less effective in protecting XG4; greater concentrations interfered with the RNA ligase activity (data not shown). Electrophoretically purified L2(1)M forms of XF1, XF3, and XG5, but not XG4, were converted efficiently to C2(1)M (Fig. 6B), in a reaction dependent on addition of rATP. These results suggest that ligation of XF3, XG4, and XG5 L2(1)M to C2(1)M might also be detected in protoplasts if sufficient expression could be achieved. In order to increase the extent of transcription, the construction in pAC1.3TTSC was modified by insertion of a tRNA-derived PolIII promoter to the 59 side of the subdimeric XF3 sequence (Bourque and Folk, 1992; Murfett et al., 1995; Perriman et al., 1995). Transient expression of this construction allowed a reproducible assessment of L2(1)M conversion to C2(1)M (Fig. 6C), which for XF1, XF3, and XG5, but not XG4, was greater than that usually observed for wild-type L2(1)M. Results from the 127 nucleotide residue mutant XF-3 (Fig. 7B) construction showed that secondary structure regions B, C, D, E, F, and G (Fig. 1A; Table 1) are dispensable for the ligation reaction that generates C2(1)M in protoplasts and in wheat germ extract. sTRSV RNA mutant constructions XG4 and XG5 were designed to test further the possible role of stem H (Fig. 1A) but without altering nucleotide sequence 355±358, which is required for the self-cleavage reaction (Fig. 1B). In XF3, nucleotide residues ACG 262± 264 form part of truncated stem H by base pairing to residues UGU 356±358, including the G264±U356 wobble pair. In XG4, UGC 262±264 replaces the ACG 262±264 of XF3, presumably disrupting all three base

SATELLITE RNA CIRCLES

421

TABLE 1 Altered Regions in Calculated Secondary Structures of sTRSV RNA Mutants sTRSV RNA mutant

a

Altered regions of calculated secondary structureb

D-75 (D227±247) D-17 79U CGGAU CCGCGUAAACU 80A D-99 (D56±92) D-99/16 (D56±164) D-16 (D150±164) D-17/43 (D80±164) D-94 (D121±133) D-101 (D161±186)c D-88 (D281±298) D-25 (D274±280) D-25/22 (D274±299) D-9 (D294±318) D-60 (D188±224)c D-47 (D261±271)c D-22 (D242±299)c D-22/47 (D242±271) D-79 (D311±344)c D-22/25 (D242±280)c D-47/22 (D261±299)c

I

Exceptions out of 19d

5

XF1 (D53±211) C52 GGGUGUC G212 XF2 (D53±211) C52 GGGUGUC G212 (D274±349) U273 AG A350 XF3 (D53±211) C52 GGGUGUC G212 (D268±350) G267 CGG U351

A A A A

I I I

B B

C C C C C C

A A A A A A

D

D

D

7

E E E E E E [E] E E E [E]

F F F F F F F F F F

G G G G G G G G G G

h* H H H H H H H

6

5

4

2

9

11

6

[B]

[C]

[D]

[B]

[C]

[D]

[E]

[F ]

[G]

[B]

[C]

[D]

[E]

[F ]

[G]

h*

a

Sequence numbering corresponds to that of wild-type sTRSV RNA (Fig. 1A). Mutant D-17 retains all of the wild-type sequence and has the specified sequence inserted between nucleotide residues 79 and 80. Other mutants of the D-series are defined by a deletion, in parentheses, which is replaced by CGGAUCCG. XF-1, XF-2, and XF-3 have the extensive deletions and replacement indicated. b Fig. 1A defines the altered regions of the secondary structure identified by letter. Underline indicates that a sequence composing part of the wild-type secondary structure is altered in the mutant. Brackets define structures for which the entire sequence was deleted. h* identifies a H helix that is intact except for truncation by one (D-9) or three (XF3) base pairs (Figs. 1C and 7B). c Indicates mutants for which circle formation was reduced significantly during transient expression from an ACMV vector (Fig. 3B, groups b, c, and d; Fig. 5B, D-22 only) d For mutants listed above this line, the number entered on this line is the sum of those mutants for which (i) an altered region of calculated secondary structure is not associated with reduced C2(1)M accumulation and (ii) an unaltered region (with entries h* being considered as unaltered H) is associated with diminished C2(1)M accumulation.

pairs. The resulting computed secondary structure is unlike that of XF3 (Figs. 7C and 7B). In compensatory mutant construction XG5, GCA 262±264 replaces the ACG 262±264 of XF3, restoring the truncated stem H in the computed structure (Fig. 7D), with one wobble base pair but without using the XF3 trinucleotide sequence. In both wheat germ extract (Fig. 6B, lanes 5 and 6) and after transient expression in protoplasts (Fig. 6C, lane 5), XG4 C2(1)M accumulation was reduced substantially compared to that observed for XF3, although the ligation reaction was not eliminated. As expected, the XG5 construction showed restoration of ligation, with accumulation of C2(1)M comparable to that for XF3 in both systems (Fig. 6B, lanes 7 and 8; Fig. 6C, lane 6).

DISCUSSION We presume that the first step in the replication of sTRSV RNA, as initiated by L2(1)M molecules, is the formation of C2(1)M and that this reaction is catalyzed by a cell-encoded RNA ligase. C2(1)M was found to be as effective as L2(1)M at initiating replication of sTRSV RNA (Buzayan et al., 1995). Conversion of L2(1)M to C2(1)M was observed in protoplasts and plants inoculated with satellite RNA-expressing ACMV constructions, in the absence of infecting TRSV. Tobacco transgenically expressing sTRSV RNA previously also was found to accumulate C2(1)M (Robaglia et al., 1993). For the wildtype L2(1)M-expressing construction investigated here, the proportions of C2(1)M and L2(1)M (Figs. 4 and 5B)

422

CHAY, GUAN, AND BRUENING

were similar to those observed in extracts of tissue infected with TRSV and sTRSV RNA (Passmore and Bruening, 1993), i.e., roughly equimolar amounts. Previous results showed that the bulk of the C2(1)M electrophoretically purified from extracts remained circular when incubated under conditions favorable to self-cleavage of dimeric sTRSV(1)RNA (Passmore and Bruening, 1993) and has a junction phosphodiester that resisted cleavage in base (Buzayan et al., 1995). Therefore, the C2(1)M extracted from protoplasts likely has the unit length sTRSV RNA ligated to form a 39,59-phosphodiester, 29-phosphomonoester, as was detected in two circular RNAs, those of Solanum nodiflorum mottle sobemovirus and velvet tobacco mottle sobemovirus (Kiberstis et al., 1985) and is characteristic of RNA ligated in vitro after incubation with rATP in wheat germ extract (Konarska et al., 1982; Schwartz et al., 1983). However, whether such C2(1)M serves as template for rolling circle transcription in sTRSV RNA replication remains unknown. The ACMV vector effected expression of wild-type and mutant sTRSV(1)RNAs similarly, as judged by amounts of accumulated RNA for molecules as small as the 207nucleotide residue XF1. However, the 127 nucleotide residue XF3, XG4, and XG5 molecules were difficult to detect using the vector pAC1.3TTSC. Two possible explanations for reduced accumulation of the small sTRSV(1)RNA molecules is that they are unstable when expressed in protoplasts or that their small size is incompatible with efficient expression from the ACMV vector without a Pol III promoter (Perriman et al., 1995). XG4 nearly disappeared when incubated in wheat germ extract under conditions that XF1, XF3, and XG5 survived, and the only condition found that preserved XG4, in vitro, without appreciably interfering with ligase activity was addition of a carefully calibrated concentration of vanadyl nucleosides (Fig. 6B, lanes 3±8). Reproducible accumulations in protoplasts of L2(1)M and C2(1)M for XF3, XG4, and XG5 (Fig. 6B, lane 5 and 6) were obtained after modifying the ACMV vector by incorporation of an RNA polymerase III promoter, which presumably enhanced transcription sufficiently to overcome losses due to instability of the RNAs, particularly XG4. Mutants with deletions in three nucleotide sequence regions of the sTRSV(1)RNA molecule showed reduced conversion of L2(1)M to C2(1)M (Fig. 3). The sequence regions and corresponding mutants are: (1) 161±224, D-101, and D-60; (2) 242±299, D-22/25, D-22, D-47, and D-47/22; (3) 311±344, D-79. The predicted folding of sTRSV(1)RNA (Fig. 1A) is a compact structure with only a few nucleotide residues not involved in stem loops, albeit imperfect stem loops. We relied on the structure shown in Fig. 1A as a guide for preparing more extensive deletions of the sTRSV RNA sequence. The mutants D-101 and D-60 have mutations mapping to sequences that form calculated secondary structure region D and

FIG. 6. Comparison of circle accumulation, in vitro and in vivo, for a set of extensively deleted sTRSV RNA mutants. (A) Diagram of deletion mutants XF1 and doubly deleted XF3, XG4, and XG5. Sequences of these mutants are presented in Fig. 7. (B) 32P-labeled, electrophoretically purified L2(1)M sTRSV RNA mutants, as indicated above the paired lanes, were incubated with wheat germ S-100 extract without (odd-numbered lanes) or with (even numbered lanes) rATP before analysis by electrophoresis and autoradiography. Reaction mixtures except those for XF1 included 0.3 mM vanadyl ribonucleosides. (C) Approximately 106 protoplasts were electroporated with buffer alone (lane 3) or approximately 10 mg of ACMV plasmid vector bearing sequences of wild-type sTRSV RNA (T, lane 1) or mutant XF1 (lane 2). For the set of mutants XF3 (lane 4), XG4 (lane 5), and XG5 (lane 6), the ACMV vector included a tRNA sequence to enhance transcription. Analyses were as in Fig. 2E. Upper and lower horizontal lines beside the autoradiograms locate migration positions for C2(1)M and L2(1)M bands, respectively, based on the mobilities of marker RNAs prepared as in (B). Fractions below the lanes in B and C are the radioactivities associated with the bands, expressed as C2(1)M/ [L2(1)M1, C2(1)M]. In (C) results from three experiments are presented as the average and the range.

parts of regions C and A of the wild-type sTRSV RNA (Fig. 1A). However, among the 19 D-series mutants tested for correlation between calculated secondary structure alterations and reduced circle formation, the correlation was poorest for calculated secondary structure regions B and C and low for D (Table 1, line 20; Fig. 1A), suggesting that these regions are not important to the ligation reaction. Therefore, we prepared mutant XF1, from which is deleted the contiguous sequence that forms regions B, C, and D. This substantial deletion did not

SATELLITE RNA CIRCLES

423

FIG. 7. Computed secondary structures of sTRSV RNA mutants with extensive deletions. Numbered nucleotide residues correspond to the wild-type sequence (Fig. 1A). Secondary structure regions labeled as described in the legends to Fig. 1A and Table 1. (A) The XF1 secondary structure corresponds to that of intact sTRSV RNA except that the right hand three imperfect stem-loops (Fig. 1A, structures B, C, and D) and adjacent A-U base pair (nucleotide residues 53±211, Fig. 6A) are replaced by the terminally G-C base-paired loop GGGUGUC. (B) The XF3 sequence and secondary structure are those of XF1 with replacement of the left hand three imperfect stem-loops [Fig. 1A, structures E, F, G, and part of H, nucleotide residues 268±350 (Fig. 6A)] by CGG. (C) For mutant XG4, the bracketed trinucleotide sequence ACG of XF3 (nucleotide residues 262±264) is replaced by UGC, and (D) for XG5 the trinucleotide sequence is replaced by GCA. The computed folding of XG5 (only the left-most stem-loop shown) corresponds to that of XF3, whereas the computed folding of XG4 is distinct.

diminished circle formation in vitro or in vivo (Fig. 6B, lane 2, and Fig. 6C, lanes 1 and 2), suggesting that the nucleotide sequences deleted in D-101 and the 59-most region of D-60, specifically, 188±210, are not important in circle formation. Similarly, mutants D-22 and D-47/22 delete the entire sequence that encompasses secondary structure region E. These two mutants showed the greatest reduction in circle formation among the D-series mutants tested (Fig. 3B). However, alterations in computed folding for the sequences of regions E, F, and G (Fig. 1A) were not well correlated with reductions in circle formation (line 20 of Table 1). Therefore, the corresponding contiguous sequence was deleted to form mutant XF3. XF3 L2(1)M was converted to C2(1)M to an extent comparable or

greater than that for XF1. Hence, the reductions in circle formation seen for D-22, D-22/25, D-47/22, and D-79 relative to wild-type sTRSV RNA (Figs. 3B and 5B) are unlikely to have resulted from the direct effects of deleting sequences within the range 271±348, corresponding to regions E, F, and G. Sequences affecting C2(1)M accumulation may reside within nucleotide residues 1±45 and 355±359 (Figs. 1A and 1B), but these sequences, are not readily susceptible to testing by our approach, because our approach requires that the self-cleaving hammerhead sequence (Forster and Symons, 1987) remain intact to generate L2(1)M from the ACMV vector-expressed subdimeric sequence. The entire computed secondary structure region I and part of region A are derived from

424

CHAY, GUAN, AND BRUENING

nucleotide residues 1±45. Mutants D-75, D-22/47, D-60, and D-22 have deleted sequences that contribute to structure A in the wild-type sequence. D-75, D-22/47 form circles efficiently but D-60 and D-22 do not. For both structures I and A, the correlation between disruption of the computed secondary structure and reduced circle formation is not strong (Table 1, line 20), providing indirect evidence against an essential contribution of structures I and A to C2(1)M formation. The best correlation between reduced circle formation and a predicted region of folding is that for helix H (Fig. 1A), with only two exceptions. No alteration of helix H is predicted for D-101 (Fig. 1C), but circle formation was reduced for this mutant. D-22/47 had undiminished circle formation but helix H is substantially altered in the calculated folded structure (Fig. 1C). Observations for the substitution mutants XG4, which shows reduced circle formation and no predicted helix H, and for XG5, with undiminished circle formation and intact truncated helix H, support the notion that the presence of helix H makes the corresponding L2(1)M a better substrate for an RNA ligase in tobacco protoplasts. Predicted secondary structures for two other small satellite RNAs of nepoviruses also show the 39 end incorporated into an imperfect stem (Kaper et al., 1988; Rubino et al., 1990). The experiments reported here can be considered to test the substrate specificity of an enzyme in vivo, by submitting different members of the D-series L2(1)M RNAs to the same ligase enzyme in ACMV vector-transfected protoplasts. A presumed natural substrate for wheat germ RNA ligase in the splicing of tRNA precursors, i.e., paired tRNA half molecules (Stange et al., 1992; Stange et al., 1988), does not obviously resemble XF3 (Fig. 7B) in secondary structure. In contrast to yeast tRNA ligase, which shows specificity for tRNA half molecules (Apostol and Greer, 1991; Greer et al., 1983), the wheat germ activity is able to ligate a variety of substrates, including oligoribonucleotides such as (Up)nG.p and the V 59-terminal fragment of TMV RNA, excised intervening sequences, potato spindle tuber viroid RNA, and sTRSV RNA (Branch et al., 1982; Buzayan et al., 1995; Schwartz et al., 1983), although presumably not with the same efficiency. Therefore, it is not surprising that even XG4, with a predicted secondary structure (Fig. 7C) radically different from those of XF3 and XG5, should be ligated to a significant, but much reduced, extent (Fig. 6B, lane 6, Fig. 6C, lane 5), compared to XF3 and XG5. We did not observe a perfect correlation between the loss of specific calculated secondary structures and reduced C2(1)M formation, which would have corresponded to one or more entries of 0 in line 20 of Table 1. However, the predicted secondary structures were in general a very reliable guide to choosing boundaries for deleting sequences such that the function of C2(1)M formation was retained. It is possible that, when extensive secondary structure is predicted for a sequence, as

is the case for sTRSV RNA, the result has greater validity than when more limited secondary structure is predicted. ACKNOWLEDGMENTS We are grateful to John Stanley of the John Innes Centre, Norwich, UK, for gifts of ACMV-derived plasmids, to James Haseloff and Wayne Gerlach for plasmid pTTS-B and its derivatives, encoding the D-series sTRSV RNA mutants, and to Rhonda Perriman for pGtRNA. Paul Feldstein transferred the ACMV inserts from M13 constructions to plasmid vectors, which made our initial experiments possible. Research reported here was supported by the Competitive Grants Program of the Cooperative State Research Service, United States Department of Agriculture, under Grant NRICGP 91-37303-6695, and by the Agricultural Experiment Station of the University of California.

REFERENCES Apostol, B. L., and Greer, C. L. (1991). Preferential binding of yeast transfer RNA ligase to precursor transfer RNA substrates. Nucleic Acids Res. 19, 1853±1860. Bourque, J. E., and Folk, W. R. (1992). Suppression of gene expression in plant cells utilizing antisense sequences transcribed by RNA polymerase III. Plant Mol. Biol. 19, 641±647. Branch, A. D., Robertson, H. D., Greer, C., Gegenheimer, P., Peebles, C., and Abelson, J. (1982). Cell-free circularization of viroid progeny RNA by an RNA ligase from wheat germ. Science 217, 1147±1149. Bruening, G. (1990). Replication of satellite RNA of tobacco ringspot virus. Semin. Virol. 1, 127±134. Bruening, G., Buzayan, J. M., Hampel, A., and Gerlach, W. L. (1988). Replication of small satellite RNAs and viroids: Possible participation of non-enzymatic reaction. In ``RNA Genetics: Retroviruses, Viroids, and RNA Recombination'' (E. Domingo, J. J. Holland, and P. Ahlquist, Eds.), Vol. II. CRC Press, Boca Raton, FL. Bruening, G., Passmore, B. K., Van Tol, H., Buzayan, J. M., and Feldstein, P. A. (1991). Replication of A Plant Virus Satellite RNA Evidence Favors Transcription of Circular Templates of Both Polarities. Mol. Plant±Microbe Int. 4, 219±225. Buzayan, J. M., Feldstein, P. A., Segrelles, C., and Bruening, G. (1988). Autolytic processing of a phosphorothioate diester bond. Nucleic Acids Res. 16, 4009±4024. Buzayan, J. M., Gerlach, W. L., and Bruening, G. (1986b). Spontaneous ligation of RNA fragments with sequences that are complementary to a plant virus satellite RNA. Nature (London) 323, 349±353. Buzayan, J. M., Gerlach, W. L., Bruening, G., Keese, P., and Gould, A. a. V., 186±199. (1986a). Nucleotide sequence of satellite tobacco ringspot virus RNA and its relationship to multimeric forms. Virology 151, 186±199. Buzayan, J. M., Van Tol, H., Zalloua, P. A., and Bruening, G. (1995). Increase of satellite tobacco ringspot virus RNA initiated by inoculating circular RNA. Virology 208, 832±837. Chomczynski, P. (1993). A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 15, 532±534, 536±536. Church, G. M., and Gilbert, W. (1984). Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, 1991±1995. Davanloo, P., Rosenberg, A. H., Dunn, J. J., and Studier, F. W. (1984). Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 81, 2035±2039. Devereux, J., Haeberli, P., and Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387±395. Feldstein, P. A., and Bruening, G. (1993). Catalytically Active Geometry in the Reversible Circularization of Mini-Monomer Rnas Derived from the Complementary Strand of Tobacco Ringspot Virus Satellite Rna. Nucleic Acids Res. 21, 1991±1998.

SATELLITE RNA CIRCLES Feldstein, P. A., Buzayan, J. M., and Bruening, G. (1989). Two sequences participating in the autolytic processing of satellite tobacco ringspot virus complementary RNA. Gene (Amsterdam) 82, 53±62. Forster, A. C., and Symons, R. H. (1987). Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site. Cell 50, 532±534, 536±536. Gerlach, W. L., Llewellyn, D., and Haseloff, J. (1987). Construction of a plant disease resistance gene from the satellite RNA of tobacco ringspot virus. Nature (London) 328, 802±805. Greer, C. L., Peebles, C. L., Gegenheimer, P., and Abelson, J. (1983). Mechanism of action of a yeast RNA ligase in tRNA splicing. Cell 32, 537±546. Haseloff, J., and Gerlach, W. L. (1988). Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature (London) 334, 585±591. Haseloff, J., and Gerlach, W. L. (1989). Sequences required for selfcatalyzed cleavage of the satellite RNA of tobacco ringspot virus. Gene (Amsterdam) 82, 43±52. Hertel, K. J., Pardi, A., Uhlembeck, O. C., Koizumi, M., Ohtsuka, E., Uesugi, S., Cedergren, R., Eckstein, F., Gerlach, W. L., Hodgson, R., and Symons, R. H. (1992). Numbering system for the hammerhead. Nucleic Acids Res. 20, 3252. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene (Amsterdam) 77, 51±60. Kaper, J. M., Tousignant, M. E., and Steger, G. (1988). Nucleotide sequence predicts circularity and self-cleavage of 300-ribonucleotide satellite of arabis mosaic virus. Biochem. Biophys. Res. Commun. 154, 318±325. Kiberstis, P. A., Haseloff, J., and Zimmern, D. (1985). 29 Phosphomonoester, 39-59 phosphodiester bond at a unique site in a circular viral RNA. EMBO J. 4, 817±827. Kiefer, M. C., Daubert, S. D., Schneider, I. R., and Bruening, G. (1982). Multimeric forms of satellite tobacco ringspot virus RNA. Virology 137, 371±381. Konarska, M., Filipowicz, W., and Gross, H. J. (1982). RNA ligation via 29-phosphomonoester, 39,59-phosphodiester linkage: Requirement of 29,39-cyclic phosphate termini and involvement of a 59-hydroxyl polynucleotide kinase. Proc. Nat. Acad. Sci. USA 79, 1474±1478. Linthorst, H. J. M., and Kaper, J. M. (1984). Circular satellite-RNA molecules in satellite of tobacco ringspot virus infected tissue. Virology 137, 206±210. Murfett, J., Bourque, J. E., and McClure, B. A. (1995). Antisense suppression of S-RNase expression in Nicotiana using RNA polymerase II- and III-transcribed gene constructs. Plant Mol. Biol. 29, 201±212. Owens, R. A., and Schneider, I. R. (1977). Satellite of tobacco ringspot virus RNA lacks detectable mRNA activity. Virology 80, 222±224. Passmore, B. K., and Bruening, G. (1993). Similar structure and reactivity of satellite tobacco ringspot virus RNA obtained from infected tissue and by in vitro transcription. Virology 197, 108±115. Passmore, B. K., Van Tol, H., Buzayan, J. M., Stabinsky, D., and Bruening, G. (1995). Trace amount of satellite RNA associated with tobacco ringspot virus: Increase stimulated by nonaccumulating satellite RNA mutants. Virology 209, 470±479. Perriman, R., Bruening, G., Dennis, E. S., and Peacock, W. J. (1995). Effective ribozyme delivery in plant cells. Proc. Natl. Acad. Sci. USA 92, 6175±6179. Ponz, R., Rowhani, A., Mircetich, S. M., and Bruening, G. (1987). Cherry leaf roll virus infections are affected by a satellite RNA that the virus does not support. Virology 160, 183±190. Prody, G. A., Bakos, J. T., Buzayan, J. L., Schneider, I. R., and Bruening, G. S. (1986). Autolytic processing of dimeric plant virus satellite RNA. Science 321, 1577±1580.

425

Robaglia, C., Bruening, G., Haseloff, J., and Gerlach, W. L. (1993). Evolution and replication of tobacco ringspot virus satellite RNA mutants. EMBO J. 12, 2969±2976. Rubino, L., Tousignant, M. E., Steger, G., and Kaper, J. M. (1990). Nucleotide sequence and structural analysis of two satellite RNAs associated with chicory yellow mottle virus. J. Gen. Virol. 71,(Pt 9), 1897±1903. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). ``Molecular Cloning: A Laboratory Manual,'' 2nd ed., 1, 2, 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Schneider, I. R. (1969). Satellite-like particle of tobacco ringspot virus that resembles tobacco ringspot virus. Science 166, 1627±1629. Schneider, I. R. (1971). Characteristics of the satellite-like virus of tobacco ringspot virus. Virology 45, 108±112. Schneider, I. R. (1977). Defective plant viruses. In ``Virology in Agriculture'' (T. O. Diener, Ed.), Vol. I, pp. 201±219. Allenheld, Osmun Universe Books, Beltsville, MD. Schneider, I. R., Hull, R., and Markham, R. (1972). Multidense satellite of tobacco ringspot virus: a regular series of components of different densities. Virology 47, 320±330. Schwartz, R. C., Greer, C. L., Gegenheimer, P., and Abelson, J. (1983). Enzymatic mechanism of an RNA ligase from wheat germ. J. Biol. Chem. 258, 8374±8383. Sheldon, C. C., and Symons, R. H. (1993). Is hammerhead self-cleavage involved in the replication of a virusoid in-vivo? Virology 194, 463± 474. Sogo, J. M., and Schneider, I. R. (1982). Electron microscopy of doublestranded nucleic acids found in tissue infected with the satellite of tobacco ringspot virus. Virology 117, 401±415. Stange, N., Beier, D., and Beier, H. (1992). Intron excision from tRNA precursors by plant splicing endonuclease requires unique features of the mature tRNA domain. Eur. J. Biochem. 210, 193±203. Stange, N., and Beier, H. (1986). A gene for the major cytoplasmic tRNA Tyr from Nicotiana rustica contains a 13 nucleotides long intron. Nucleic Acids Res. 14, 8691. Stange, N., Gross, H. J., and Beier, H. (1988). Wheat germ splicing endonuclease is highly specific for plant pre-tRNAs. Eur. Mol. Biol. Org. J. 7, 3823±3828. Stanley, J. (1983). Infectivity of the cloned geminivirus genome requires sequences from both DNAs. Nature (London) 305, 643±645. Stanley, J., and Gay, M. (1983). Nucleotide sequence of cassava latent virus DNA. Nature (London) 301, 200±202. Symons, R. H. (1992). Small catalytic RNAs. Annu. Rev. Biochem. 61, 641±671. Symons, R. H., Haseloff, J., Visvader, J. E., Keese, P., Murphy, P. J., Gordon, K. H. J., and Bruening, G. (1985). On the mechanism of replication of viroids, virusoids, and satellite RNAs. In ``Survival Pathogens of Plants and Animals: Viroids and Prions'' (K. Maramorosch, and J. J. McKelvey, Jr., Eds.), pp. 235±263. Academic Press, New York. Van Tol, H., Buzayan, J. M., and Bruening, G. (1991). Evidence for spontaneous circle formation in the replication of the satellite RNA of tobacco ringspot virus. Virology 180, 23±30. Van Tol, H., Buzayan, J. M., Feldstein, P. A., Eckstein, F., and Bruening, G. (1990). Two autolytic processing reactions of a satellite RNA proceed with inversion of configuration. Nucleic Acids Res. 18, 1971± 1976. Ward, A., Etessami, P., and Stanley, J. (1988). Expression of a bacterial gene in plants mediated by infectious geminivirus DNA. EMBO J. 7, 1583±1588.