Genetic RNA recombination is a process that joins together two non-contiguous RNA segments (King, 1988; Lai, 1992) . The term currently is used only for RNA molecules that carry genomic functions (i.e. genomes of RNA viruses, satellites and viroids), thereby excluding RNA processing, splicing and editing. RNA recombination is well documented for a large number of viruses, and is believed to have affected viral evolution and adaptation (King, 1988; Strauss and Strauss, 1988; Lai, 1992; Dolja and Carrington, 1992; Simon and Bujarski, 1994) . Recent studies on RNA recombination with RNA viruses revealed roles for RNA recombination in creating novel chimeric viruses, 2392 © Oxford University Press recovering functional genomes from mutated (damaged) RNAs and contributing to the quasispecies nature of RNA viruses (Zimmern, 1988; Lai, 1992; Nagy and Bujarski, 1996; Simon and Nagy, 1996) . RNA recombination is also a risk factor in some transgenic systems by facilitating the generation of unwanted viral recombinants (Greene and Allison, 1994) .
Early studies on RNA recombination suggested an infrequent occurrence and a scattered distribution of junction sites (Kirkegaard and Baltimore, 1986; King, 1988) . These features and the existence of strong selection pressure favoring only the accumulation of viable recombinants made studies on the molecular mechanisms of RNA recombination difficult. However, comparison of RNA recombinants isolated in numerous viral systems led to the classification of recombinants as homologous or non-homologous (King, 1988; Lai, 1992) . The former recombinants were those that were generated from two homologous RNAs and had junction sites located within similar sequences, while the latter were derived from two dissimilar RNAs.
Recent development of site-(or short region-) specific, targeted RNA recombination systems for brome mosaic bromovirus (BMV), tomato bushy stunt tombusvirus and turnip crinkle carmovirus (TCV) allowed further refinement of the RNA determinants and molecular mechanisms of RNA recombination (reviewed by . For the tricomponent BMV, inserting a short RNA1-derived sequence into an RNA3 derivative in the complementary orientation induced the generation of nonhomologous RNA1-RNA3 recombinants (Nagy and Bujarski, 1993) . In addition, insertion of a short RNA2derived region in the direct orientation into an RNA3 derivative facilitated RNA2-RNA3 crossovers that were of the homologous type, since the junctions were located precisely within the common regions . A replicase-driven template-switching mechanism for both homologous and non-homologous recombination was supported by the distribution of recombinant junctions on the RNAs and the observation that mutations within the two virus-encoded components of the replicase influenced the frequency of recombinant accumulation and the distribution of junction sites Bujarski, 1993, 1997; Figlerowitz et al., 1997) . Using chimeric tombusviruses, White and Morris (1995) found that preferred junction sites were the 5Ј termini and strong hairpin structures in the donor templates. Hairpin structures that were proposed to facilitate the re-initiation of RNA synthesis during the template-switching events were also involved in generating TCV recombinants (Cascone et al., 1990 (Cascone et al., , 1993 Carpenter et al., 1995) .
TCV is a 4054 base RNA virus that uniquely is associated with a number of subviral RNAs, including satellite, defective-interfering and chimeric RNAs (Simon and Nagy, 1996) . RNA species in the latter two groups are the products of natural recombination. For example, the chimeric sat-RNA C (356 nucleotides) is composed of sequences similar to a satellite RNA 194 nucleotides) at its 5Ј end and two 3Ј-proximal regions from TCV genomic RNA at its 3Ј end (Simon and Howell, 1986) . High frequency recombination was observed in vivo between sat-RNA D and sat-RNA C, with the recombination junctions clustered close to or at the 3Ј end of sat-RNA D and at the base of a hairpin (designated motif1hairpin) in the center portion of sat-RNA C (Cascone et al., 1990 (Cascone et al., , 1993 . Non-compensatory mutations in the stem of the motif1-hairpin eliminated detectable recombinants in vivo. In addition, part of the sequence of the motif1-hairpin contains similarity with the 5Ј-end region of the TCV genomic RNA that probably contains the promoter for positive strand synthesis. These results suggested that the motif1-hairpin is involved in recruitment of the RdRp during re-initiation of synthesis following the template switch. The model predicted that the viral replicase (RNA-dependent RNA polymerase, RdRp) uses the nascent, positive-stranded sat-RNA D as a primer to resume RNA elongation on the acceptor sat-RNA C template during the recombination events (Cascone et al., 1993; Simon and Nagy, 1996) .
The replicase-mediated template-switching models suggest that recombination can be divided into three steps: (i) generation of the primer on the donor RNA; (ii) strand transfer and the binding of the RdRp to the acceptor RNA; and (iii) primer elongation on the acceptor RNA (Jarvis and Kirkegaard, 1991; Lai, 1992; . In this report, two questions relevant to the strand transfer and primer elongation steps have been studied: (i) how a promoter-dependent RdRp, such as the TCV RdRp, associates with the acceptor RNAs and (ii) how the RdRp is capable of primer extension, a process fundamentally different from de novo initiation. We report the development of a cell-free (in vitro) system that uses hybrid RNAs that resembled putative recombination intermediates to program partially purified TCV RdRp preparations. These hybrid RNAs supported 3Ј-terminal extension (3Ј-TX), which is analogous to primer extension proposed to occur in vivo by the TCV RdRp (Cascone et al., 1993; Simon and Nagy, 1996) . Comparison of the in vitro and in vivo results demonstrates that the in vitro system can faithfully copy most of the aspects of the in vivo system, giving direct support for a replicase-driven template-switching mechanism for RNA recombination.
The TCV RdRp, similarly to many other viral RdRps, replicates the TCV genomic RNA and all the associated RNAs by recognizing specific promoter sequences/structures present on these RNAs and initiating RNA synthesis de novo Simon, 1994, 1995; Guan et al., 1997) . In addition, the TCV RdRp is capable of primer elongation, but only from the 3Ј end of the plus strand and only in the presence of a functional promoter (i.e. the TCV RdRp was unable to extend primers hybridizing randomly at internal positions) ; C.Song and A.E. Simon, unpublished) . However, according to the model of RNA recombination in TCV, the RdRp must be able to extend on the nascent strand at internal positions on the template RNA using the nascent RNA strand as a primer during the crossover events. To determine whether the TCV RdRp is capable of primer extension when supplied with putative recombination intermediates, we have used an in vitro system that includes the putative recombination intermediates of a previously defined RNA recombination system between sat-RNA C and sat-RNA D: the negative-stranded sat-RNA C, TCV RdRp, and positive-stranded, nearly full-length nascent strand of sat-RNA D ( Figure 1A ). The model also predicts that the nascent positive strand of sat-RNA D is used by the TCV RdRp as a primer to extend RNA synthesis on the acceptor sat-RNA C from the base of the motif1-hairpin (Cascone et al., 1990) . Sequence comparison of junction regions revealed complementarity between the 3Ј-end region of the nascent sat-RNA D positive strand and a region 3Ј of the motif1-hairpin in the negative strand of sat-RNA C ( Figure 1A) , with the sequences predicted to form a stable base-paired structure (priming stem).
The cell-free (in vitro) system makes use of a partially purified, template-dependent TCV RdRp preparation (Song and Simon, 1994) . The TCV RdRp preparation was programed with RNA templates that were designed to resemble the above-described putative recombination intermediates ( Figure 1B ). One of these constructs, designated CD-13mini, consisted of sequences corresponding to the central portion of the negative strand of sat-RNA C (total of 84 nucleotides, including the in vivo recombination hotspot region and the motif1-hairpin, plus short 5Ј-and 3Ј-flanking sequences) and a 28 nucleotide segment of the positive-stranded sat-RNA D with the 3Ј terminus representing the in vivo recombination hotspot position -13 (relative to the 3Ј end) ( Figure 1B ). This hybrid construct was designed to avoid known transcriptional promoters in sat-RNA C minus strands and sat-RNA D plus strands. A six-nucleotide artificial loop sequence was used to covalently link the sat-RNA D plus strand fragment to the sat-RNA C minus strand fragment, as depicted in Figure 1B , to ensure that an optimal 1:1 molar ratio of interacting sequences were in proximity and in the correct orientation. If the model for recombination between sat-RNAs D and C is correct, then the TCV RdRp should start 3Ј-TX, analogously to recombination in vivo, at the base of the motif1-hairpin in the hybrid construct ( Figure 1C ). This 'self-primed' 3Ј-TX reaction is expected to generate a product that has a hairpin-like structure with a long double-stranded region and short loop sequence (schematically shown in Figure 1C ).
The major 3Ј-TX product generated using CD-13mini template in the RdRp reaction migrated faster than the 117 nucleotide full-length input RNA in denaturing polyacrylamide gels, as expected for a hairpin-like RNA molecule possessing an extremely stable secondary structure. Treatment of the 3Ј-TX product with single strandspecific S1 nuclease resulted in degradation of the 3Ј-TX product into two new radiolabeled RNA species, one migrating at a position of~80 nucleotides, and the second at~65 nucleotides ( Figure 1D ). The occurrence of two S1 nuclease-resistant products is probably due to partial digestion at the mismatched region in the priming stem Fig. 1 . An in vitro system to study RNA recombination in TCV. (A) The in vivo system. Model for RNA recombination between sat-RNA D and sat-RNA C by a replicase-driven template-switching mechanism. The recombination intermediates are depicted, showing the possible RNA-RNA interaction between the nascent sat-RNA D plus strand, truncated at position -13 at the 3Ј end (in in vivo experiments, the junctions were located most frequently at positions -13 to -15, as counted from the 3Ј end), and sequence 3Ј of the motif1-hairpin of sat-RNA C minus strand (the acceptor RNA strand). Sat-RNA D sequence is boxed in black. Underlined nucleotides at the base of the motif1-hairpin indicate the location of the in vivo junction site hotspot in sat-RNA C. In the rectangles representing the sat-RNAs, similar regions are shaded alike. (B) In vitro system that mimics the putative in vivo recombination intermediate. Top construct (CD-13mini) contains 28 nucleotides from sat-RNA D plus strand (boxed in black), extending from position -13 at the 3Ј end, joined by a six-base artificial sequence (5Ј-GAAUUC-3Ј) to sat-RNA C minus strand sequence that includes 33 nucleotides 3Ј of the motif1-hairpin, the motif1-hairpin, and 18 nucleotides 5Ј of the hairpin (plus two non-viral G residues at the 5Ј end). Bottom construct (CD-27mini), same as CD-13mini except that the sat-RNA D sequence terminates at the -27 position and two mismatches in the priming stem are mutated such that base pairing can occur throughout the priming stem. Regions referred to in the text as the priming stem, motif1-hairpin, loop and spacer are shown. (C) Products of the primer extension reactions (3Ј-TX). Addition of partially purified TCV RdRp, ribonucleotides and radioactive UTP results in an intramolecular extension from the 3Ј-terminal sat-RNA D sequence for CD-13mini and CD-27mini using sat-RNA C sequence as template. The radiolabeled portion of the product is shown by a thick dotted line. S1 nuclease-sensitive and RNase H-sensitive (following hybridization of loop1 oligo DNA to the loop region, depicted by a solid line) sites are indicated. (D) Efficient 3Ј-TX by the TCV RdRp. Denaturing gel analysis of radiolabeled 3Ј-TX products synthesized by in vitro transcription with TCV RdRp. Lanes depicted by -, S1 and H denote products that were untreated, or treated with either S1 nuclease or RNase H, respectively. M, single-stranded RNA markers (in bases). The template RNAs were CD-13mini (B), CD-14mini (one base 3Ј-end truncation derivative of CD-13mini) and CD-27mini (B). Note that in the untreated samples, the hairpin-like RNAs migrate aberrantly, much faster than their corresponding sizes in denaturing gels. of CD-13mini resulting in a shorter,~65 nucleotide product, and more complete digestion in the singlestranded loop giving the~80 nucleotide product ( Figure  1C ). Specific digestion of the loop region of the 3Ј-TX products obtained with CD-13mini template by RNase H in the presence of an oligodeoxyribonucleotide (loop1 oligo, complementary to the loop region of CD-13mini) ( Figure 1C ) resulted in a single radiolabeled RNA product migrating at a position of~85 nucleotides ( Figure 1D ). In combination with the S1 analysis, the data indicate that initiation of 3Ј-TX occurred at the base of the motif1hairpin on the CD-13mini template (schematically shown in Figure 1C ).
To investigate the role of the sat-RNA D-derived region in 3Ј-TX reactions, deletion of one base and 14 bases were made from the 3Ј end of CD-13mini ( Figure 1B) , generating CD-14mini (not shown) and CD-27mini ( Figure  1B) , respectively, which contain 3Ј ends corresponding to the -14 and -27 positions of sat-RNA D. While the -14 position was a frequent junction in in vivo recombinants, the -27 position has never been found (Cascone et al., 1990 (Cascone et al., , 1993 Carpenter et al., 1995) . In addition, CD-27mini contained two mutations that allowed for complete hybridization between sat-RNA D and the region 3Ј of the sat-RNA C motif1-hairpin ( Figure 1B ). CD-14mini and CD-27mini generated the hairpin-sized 3Ј-TX products that migrated similarly to the 3Ј-TX product obtained with CD-13mini ( Figure 1D ). The sizes of the S1 nucleasedigested products were also similar for CD-13mini and CD-14mini, while CD-27mini had only a single S1 nuclease-resistant product that co-migrated with the~80 nucleotide product for CD-13mini ( Figure 1D ). The existence of a single S1 nuclease-resistant radiolabeled 3Ј-TX product for CD-27mini was consistent with the lack of mismatched bases in the primer stem region of CD-27mini eliminating the S1 nuclease-sensitive site present in CD-13mini and CD-14mini. Since CD-13mini, CD-14mini and CD-27mini generated single RNase H-resistant radiolabeled products of similar size, 3Ј-TX for CD-14mini and CD-27mini must have initiated~1 and~13 nucleotides, respectively, 3Ј of the initiation site for CD-13mini, which was at the base of the motif1-hairpin. Quantitation of the 3Ј-TX products by densitometric scanning of exposed films followed by normalization for the number of adenylate residues on the template portion of the respective RNA revealed that CD-14mini was 27.9 Ϯ 2.3% less active in generating hairpin-sized 3Ј-TX products when compared with CD-13mini. Surprisingly, CD-27mini was~6-fold (603.3 Ϯ 39.5%) more active than CD-13mini (100%) in the 3Ј-TX reactions. The elevated activity of CD-27mini was probably due to a spacer region (see Figure 1B ) located between the priming stem and motif1-hairpin and not the mutations that eliminated the mismatched bases from the priming stem, since introduction of two similar mutations to the priming stem of CD-27mini did not reduce the level of 3Ј-TX (data not shown). This CD-27mini with a mutated priming stem generated 3Ј-TX products that resulted in two radiolabeled RNA species following S1 nuclease treatment, which co-migrated with those obtained with CD-13mini (data not shown).
One of the hallmarks of recombination between sat-RNA C and sat-RNA D is the requirement for the motif1-hairpin 2395 Fig. 2 . The motif1-hairpin facilitates 3Ј-TX. (A) Sequences and structures of CD-27mini, CD-27mini/mot1, which lacks the 5Ј end sequence, and the 5Ј portion of the motif1-hairpin. The efficiency of 3Ј-TX was analyzed by denaturing PAGE, followed by autoradiography and densitometry. The data were normalized based on the amount of template-directed radioactive UTP incorporated. The standard error was calculated from three separate experiments. (B) A representative experiment showing denaturing gel analysis of radiolabeled and S1-treated 3Ј-TX products synthesized by in vitro transcription with TCV RdRp using CD-27mini, CD-27mini/mot1, GC12mini, GC12mini/mot1, AU/GCmini and AU/GCmini/mot1 (see Figure 5 ) as templates. M, single-stranded RNA markers (in bases). sequence and/or structure in vivo (Cascone et al., 1990 (Cascone et al., , 1993 ; reviewed by Simon and Nagy, 1996) . To test for involvement of the motif1-hairpin in the 3Ј-TX with our chimeric constructs in vitro, CD-27mini/mot1, which contained only the 3Ј-half of the motif1-hairpin and the priming stem, was constructed ( Figure 2A ). The 3Ј-TX product for CD-27mini/mot1 was reduced by~30-fold from the level obtained using CD-27mini ( Figure 2B ). This result demonstrates that the priming stem alone cannot support efficient 3Ј-TX and that the motif1-hairpin is required for efficient 3Ј-TX from the priming stem in vitro. Thus, as predicted by the RdRp-mediated template-switching model of recombination, 3Ј-TX (and possibly the analogous in vivo recombination reaction) is a more complex phenomenon than a simple primer extension reaction (see Discussion). In addition, the requirement for the entire motif1-hairpin in vitro suggests that the role of the motif1-hairpin may be similar in the in vitro 3Ј-TX system and the in vivo sat-RNA C-sat-RNA D recombination system.
To characterize the sequence and structural requirements of the motif1-hairpin in 3Ј-TX and recombination in vivo, three types of mutations were introduced into the hairpin: (i) destabilizing mutations that changed the stability of the hairpin; (ii) compensatory mutations that restored base pairing within the stem; and (iii) mutations in singlestranded regions that did not change the stability of the hairpin predictably. In this assay, the template was CD-14B that contains sequences similar to CD-14mini and, in addition, a 147 nucleotide sequence representing the 5Ј segment of sat-RNA C. CD-14B supported 3Ј-TX similarly to CD-14mini (data not shown).
Mutations in the motif1-hairpin of CD-14B are depicted in Figure 3B , and their effects on 3Ј-TX are shown in Table I . In addition, sat-RNA C containing the corresponding motif1-hairpin mutations were tested for in vivo RNA 2396 recombination with sat-RNA D in whole turnip plants. For these studies, the positive-stranded sat-RNA C-derived constructs contained a 22 nucleotide deletion near the 5Ј end that required repair through recombination with sat-RNA D in order to obtain viable sat-RNA C progeny in co-infections with wild-type sat-RNA D and TCV helper virus (Cascone et al., 1993) .
Stem-destabilizing mutations U183G and U185G reduced 3Ј-TX to 12.3 and 6.8%, respectively, of the level obtained with CD-14B carrying the wild-type motif1hairpin. In addition, these mutations eliminated detectable in vivo recombination between sat-RNA D and sat-RNA C (Table I) . Compensatory mutations that restored the base pairing in the motif1-hairpin increased 3Ј-TX to 21.8 and 24.4% (U183G/A206C and U185G/A202C, respectively) and restored in vivo recombination to the wild-type level. Two other stem-destabilizing mutations (LStem/3G and A189U) that reduced 3Ј-TX to 23.5 and 22.9% were recombination incompetent in vivo. Three-base compensatory mutations in constructs LStem/3GC and UStem/ 3GC that preserved the stability of the motif1-hairpin resulted in 41.0 and 25.4% 3Ј-TX activity, respectively, and showed 12 and 17% recombination activity in vivo, respectively. Deletion of the three bases in the U-bulge reduced 3Ј-TX to 9.4% and eliminated recombination in vivo (∆U3, Table I ).
Single mismatch mutations within the loop region reduced 3Ј-TX and in vivo recombination to various extents (Table I) . Mutants G191C, U192A, G194A and G195U generated 3Ј-TX products at 37.3-50.9% the level of CD-14B, while supporting in vivo recombination at 40-75% of the level of sat-RNA C with a wild-type motif1-hairpin. Among the loop mutants, those with point mutations at positions 193 and 196 (constructs C193U and U196C) inhibited both 3Ј-TX (22.0 and 27.7%, respectively) and in vivo recombination (non-detected) to the largest extent.
To characterize whether hybridization between sat-RNA D sequence and the region 3Ј of the motif1-hairpin in sat-RNA C (schematically shown as the priming stem in Figure  1B ) is required for 3Ј-TX, seven mismatch mutations that were predicted to destabilize a portion of the priming stem were introduced into CD-14B ( Figure 3A ). The resulting construct (C7) supported 3Ј-TX at only 9.9% of the level of wild-type CD-14B (Table I) . The recombinogenic, deletion version of sat-RNA C with mutations corresponding to those of C7 supported recombination in vivo at only 18% of the level of the non-mutated sat-RNA C deletion derivative (Table I) . These experiments demonstrated that sat-RNA C minus strand sequences 3Ј of the hotspot region at the base of the motif1-hairpin influenced the frequency of recombination in vivo and the efficiency of 3Ј-TX in vitro.
To test whether the sat-RNA C recombinants with mutations within their motif1-hairpin were viable, fulllength sat-RNA C constructs similar to the above in vivo tested constructs, but without the 22 nucleotide deletion, were generated and tested in whole turnip plants (for details, see Cascone et al., 1993) . All the tested full-length sat-RNA C constructs with mutations were viable (data not shown). 
The above studies demonstrate that the motif1-hairpin is required for high level 3Ј-TX. One possible role for the motif1-hairpin in 3Ј-TX is for recruitment of the RdRp.
To test whether the motif1-hairpin is able to interact with the RdRp or if the priming stem is also required, a competition assay was performed. In this assay, the template was CD-27 that contains sequences similar to CD-27mini and, in addition, a 147 nucleotide sequence representing the 5Ј segment of sat-RNA C (located 5Ј of the motif1-hairpin, as shown in Figure 4 ). The competing RNAs were derived from a variant of CD-27mini that lacked the sat-RNA D sequence, eliminating the priming stem ( Figure 4 ). The resulting Cmini series of constructs retained the wild-type motif1-hairpin (designated Cmini) or contained modifications in the motif1-hairpin. Programing the RdRp reactions with a constant amount of template (CD-27) and increasing amounts of competing RNAs (2-, 10-and 40-fold the level of template) revealed that Cmini reduced 3Ј-TX of CD-27 by 7.3% when present in 2-fold excess, by 48.0% when in 10-fold excess, and by 83.8% when in 40-fold excess (Table II) . Construct Cmini/mot1 with a partial truncation within the motif1hairpin did not compete as efficiently as Cmini, reducing 3Ј-TX of CD-27 by 5.5, 11.7 and 26.9% when present in 2-, 10-and 40-fold excess, respectively (Table II) . A derivative of Cmini with a single base alteration in the stem of the motif1-hairpin (designated Cmini/U183G) was also an inefficient competitor when compared with Cmini, reducing 3Ј-TX of CD-27 by 5.3, 21.2 and 36.1% when present in 2-, 10-and 40-fold excess. This result is consistent with the debilitating effect of U183G mutation on 3Ј-TX when present in CD-14B (see above). Addition of tRNA into the RdRp reaction in 5-, 10-and 40-fold excess, respectively, reduced 3Ј-TX of CD-27 by not more than 7% (Table II) . Overall, these data demonstrate that Cmini RNA with the wild-type motif1-hairpin, but without the priming stem, can reduce 3Ј-TX on template RNA with the primer stem. Mutated or truncated derivatives of the motif1-hairpin and the unrelated tRNA did not compete efficiently with the 3Ј-TX of the template RNA. The simplest interpretation of these results is that the wildtype motif1-hairpin competes for the RdRp, thus making less RdRp available for the 3Ј-TX reaction.
To test whether the sequence of the priming stem is important for efficient 3Ј-TX, the priming stem sequence was replaced with artificial sequences also capable of forming base-paired structures ( Figure 5A) . A 12 bp GCrich stem and seven nucleotide loop (GC12mini) was 73% as efficient at 3Ј-TX as CD-27mini. The site of initiation was at the base of the GC-rich stem, based on S1 nuclease digestion followed by size determination on denaturing gels ( Figure 5B) . A 17 bp AU-rich priming stem with one bulged nucleotide and terminating with three C residues at the 3Ј end and a seven nucleotide loop (AU/GCmini) was more efficient (129%) than CD-27mini in 3Ј-TX reactions. Constructs AU5mini and AU17mini containing only AU base-paired priming stems showed reduced 3Ј-TX activity (30.4 and 63.4%) when compared with CD-27mini ( Figure 5A ). The hairpin-like 3Ј-TX products for AU/GCmini, AU17mini and AU5mini migrated more slowly in 5% polyacrylamide-8 M urea gels, and S1 nuclease digestion revealed resistant RNA bands 12 nucleotides shorter than that obtained with CD-27mini ( Figure 5B ). Although the exact sites of initiation were not determined for these constructs, the altered migration of the 3Ј-TX products for AU/GCmini, AU5mini and AU17mini when compared with CD-27mini and GC12mini is consistent with the high AU-rich content of the priming stem, which may not be base paired during electrophoresis. In addition, the AU-rich priming stem may be cleaved by the single strand-specific S1 nuclease due to 'breathing' within the less stable AU-rich region. Alternatively, the AU-rich sequences within the priming stem may misalign, causing aberrant migration and sensitivity to S1 nuclease. To test whether the motif1-hairpin facilitates 3Ј-TX with the artificial priming stems, GC12mini/mot1 and AU/ a Average percentage and standard error of three independent 3Ј-TX analyses using CD-27 as template. See Figure 4 for a representative experiment.
GCmini/mot1, which contained only the 3Ј half of the motif1-hairpin and various priming stems, were constructed. The 3Ј-TX products for GC12mini/mot1 and AU/GCmini/mot1 were reduced by 30-fold from the level of GC12mini and AU/GCmini, respectively ( Figure 2B ), demonstrating that the priming stem alone cannot support efficient 3Ј-TX. These data demonstrate that there is no specific sequence requirement within the priming stem for 3Ј-TX in this system. The lack of sequence specificity within the priming stem suggests that the TCV RdRp does not recognize the priming stem but, instead, it recognizes the motif1-hairpin present on the template RNA.
Understanding the mechanism of RNA recombination has been hampered by the lack of in vitro systems that allow direct visualization and characterization of recombination intermediates and end-products. The available in vivo and in vitro RNA recombination systems are inefficient; thus, characterizing the recombinants requires one or more amplification steps (Lai, 1992; Nagy and Bujarski, 1992; Chetverin et al., 1997; Duggal et al., 1997; Qiao et al., 1997; Tang et al., 1997) . These post-recombination amplification steps, regardless of whether they are occurring in vivo or in vitro, have disadvantages, since they can favor amplification of some recombinants over others. For example, in most in vivo and in vitro systems (Banner and Lai, 1991; Jarvis and Kirkegaard, 1992; Nagy and Bujarski, 1992; Chetverin et al., 1997; Tang et al., 1997) , only the viable or replication-competent recombinants can be detected. In addition, the sequence of some recombinants can be modified further by mutations, deletions and rearrangements during the amplification step. Altogether, amplification can lead to misrepresentation of some recombinants in the obtained 'recombinant pool', making difficult or even excluding the possibility of drawing conclusions about the mechanism of their generation. The amplification steps can also produce artifactual recombinants and those must be discriminated carefully from 'true' recombinants. Moreover, the requirement for amplification does not allow the direct examination of recombination intermediates whose structure would be important in unraveling the mechanism of RNA recombination. The efficiency of 3Ј-TX on the template RNAs was measured as described in the legend to Figure 2. (B) Autoradiogram of denaturing PAGE of 3Ј-TX products. Lanes depicted by -and S1 denote products that were not treated, or treated by S1 nuclease, respectively. Note that in the untreated samples, the hairpin-like RNAs migrate aberrantly.
We have developed an in vitro TCV system that does not require amplification of RNA molecules. To avoid the need for amplification of the products, the in vitro system was optimized by favoring the interaction between putative RNA recombination intermediates by covalently linking the putative RNA intermediates with a short linker region. The sequence and structure of recombination intermediates were delineated from previous in vivo experiments that also resulted in a testable recombination model. This model predicted that RNA recombinants in the sat-RNA D-sat-RNA C system of TCV are formed by a replicasemediated strand transfer mechanism where the nascent strand, made on the negative-stranded donor sat-RNA D by the replicase, serves as a primer during re-initiation of RNA synthesis on the negative-stranded sat-RNA C. According to the model, this process leads to a chimeric 2399 (recombinant) sat-RNA D-sat-RNA C molecule if a putative RdRp-binding sequence (termed the motif1-hairpin) is present on the acceptor sat-RNA C. The threecomponent primer extension reaction predicted to occur in infected cells in vivo during RNA recombination should be similar to a simplified two-component 3Ј-TX reaction in vitro if the correct intermediates are in proximity in the test tube. This prediction was supported by nearly all of the data presented herein. For example, the motif1-hairpin of sat-RNA C was required for sat-RNA D-sat-RNA C recombination in vivo (Cascone et al., 1990 (Cascone et al., , 1993 this work) and also greatly facilitated 3Ј-TX in vitro. Also, the junction sites of in vivo recombinants and the initiation of 3Ј-TX products in vitro were mapped to the same region on sat-RNA C, at or close to the base of the motif1hairpin. In addition, minus strand sequences 3Ј of the motif1-hairpin of sat-RNA C greatly influenced the frequency of in vivo recombination (Cascone et al., 1993;  this work) and the efficiency of 3Ј-TX in vitro. These similarities between the in vivo and in vitro data give crucial support for the replicase-mediated template-switching model of recombination in TCV. In addition, the in vitro system allowed for defining RNA-RNA and protein-RNA interactions that probably influence template-switching events in vivo as well (see below).
In spite of the above similarities between the in vitro 3Ј-TX and in vivo recombination systems, there were exceptions that should be noted. When compared with the corresponding hairpin-destabilizing mutants, mutants with compensatory mutations in the motif1-hairpin (U183G/ A206C and U185G/A202C, Table I ) restored the efficiency of 3Ј-TX only partially, while restoring the efficiency of in vivo recombination to the wild-type level. It is possible that the mutated motif1-hairpin has a slightly different structure in vitro than in vivo. The other difference between the in vitro 3Ј-TX and the in vivo recombination systems was that the latter can be abolished completely by mutations within the motif1-hairpin, while 3Ј-TX can only be reduced to a low level, but cannot be abolished. This 'background' level of 3Ј-TX may be the consequence of inefficient interaction between the RdRp and the template RNA in the two-component 3Ј-TX system, while the possibility for interaction is reduced below the level of detection in the three-component in vivo recombination system. The amount of primer RNA (the aborted nascent sat-RNA strands) is not known in vivo, but is probably less than in the in vitro 3Ј-TX reaction. In addition, the rate of amplification or the stability of the de novo sat-RNA C-sat-RNA D recombinants can be influenced by the motif1-hairpin mutations in the in vivo recombination system, but not in our in vitro system where there is no amplification step and the RdRp products are stable hairpin-like RNAs. Other possible explanations include loss of a component by the RdRp preparations resulting in lower specificity in vitro. Finally, in the replicasedriven, strand-switching model, it is not clear whether the nascent strand together with the RdRp simply falls off the donor template and is then available for capture by the acceptor, or if the acceptor can assist in removing (i.e. can displace) the nascent strand and the RdRp from the donor. If the latter, one might expect the in vivo situation to have more stringent requirements, because the in vitro system only tests the former.
The use of an in vitro system made possible the identification of two RNA elements that can influence 3Ј-TX and, analogously, RNA recombination in TCV and its associated RNAs. The first RNA element required for the 3Ј-TX is the motif1-hairpin. Mutagenesis experiments revealed that both the sequence and the secondary structure of the motif1-hairpin can influence 3Ј-TX and in vivo recombination. Stem-destabilizing mutations in both the lower and upper stems reduced 3Ј-TX greatly and eliminated in vivo recombination. Compensatory mutations that restored the stability of the hairpin increased 3Ј-TX by 2-to 3-fold and partially restored or, for some mutants, completely restored in vivo recombination. Mutations within the loop region reduced both 3Ј-TX and in vivo recombination. Further studies will address whether there is base-specific contact(s) between the motif1-hairpin sequence and the RdRp during 3Ј-TX.
Competition studies revealed that the wild-type motif1hairpin can compete more efficiently with the 3Ј-TX of the CD-27 template than either a mutated or truncated derivative or the unrelated tRNA, suggesting that the TCV RdRp probably recognizes this element. The proposed binding of motif1-hairpin to the RdRp, however, is not strong or stable, since a 10-fold excess of competing RNA was required to produce a 50% reduction in 3Ј-TX. It is possible that the priming stem stabilizes RdRp binding to the motif1-hairpin. Alternatively, RNA synthesis (due to the 3Ј-TX) keeps the RdRp on the CD-27 template RNA for a longer period than the 'non-productive' binding to the competing RNAs that have less efficient 3Ј-TX due to the lack of a priming stem. The above experiments also demonstrated that the motif1-hairpin is not a promoter element in the strict sense, since no efficient de novo initiation was detected in the absence of the natural RdRp promoters. Accordingly, we propose that the motif1hairpin is important in recruiting the RdRp, and the complex supports efficient 3Ј-TX only if the appropriate priming stem is available.
The second RNA element required for the 3Ј-TX is the priming stem that is formed by base pairing between the 3Ј end of the RNA templates and an internal sequence 3Ј of the motif1-hairpin. Mutations that destabilized the priming stem (C7, Table I) or eliminated it by removing 3Ј-end sequence (data not shown) reduced the efficiency of 3Ј-TX. The reduced level of 3Ј-TX obtained with the above constructs was not due to changes in the primary RNA sequences, since several templates with different artificial sequences capable of priming stem formation supported efficient 3Ј-TX ( Figure 5A ). Analogously to the requirement for the priming stem for efficient 3Ј-TX, the requirement for RNA-RNA interaction is also supported by data obtained for RNA recombination in vivo between sat-RNA C and sat-RNA D, since extensive mutations (e.g. C7, Table I ) and deletions (Cascone et al., 1993) 3Ј of the motif1-hairpin on negative strands of sat-RNA C reduced the efficiency of recombination. It is important to note, however, that stable priming stems alone supported only very inefficient 3Ј-TX, arguing that the recognition of the priming stem by the TCV RdRp in the absence of the motif1-hairpin is inefficient.
As stated above, many different sequences in the priming stem can be used efficiently by the TCV RdRp if they preserve the stability of the stem. Interestingly, templates with priming stems that contained both GC and AU nucleotides were more active in 3Ј-TX than AU-or GC-rich sequences alone. Also, a 10 bp primer stem with two bulged nucleotides (AU5mini) was sufficient to support higher than background levels of 3Ј-TX. The role of short base-paired regions in promoting recombination has also been proposed for many recombination systems, including noda- (Li and Ball, 1993) , bromo- , tombusviruses (White and Morris, 1995) , Qβ (Biebricher and Luce, 1992) and Φ6 bacteriophages (Onodera et al., 1993) . In addition, sequences that contained both GC and AU nucleotides were favored for homologous recombination in BMV where RNA-RNA interaction, similar to the above priming stem in the chimeric sat-RNA D-sat-RNA C constructs, during the landing of the nascent strand on the acceptor strand has been proposed .
The priming stem in the absence of the motif1-hairpin cannot drive efficient 3Ј-TX with the TCV RdRp. Thus, the TCV RdRp, which is naturally promoter dependent and capable of de novo initiation Simon, 1994, 1995) , is different from most of the DNA-dependent DNA polymerases, reverse transcriptases and T7 DNAdependent RNA polymerase (Konarska and Sharp, 1989; Cazenave and Uhlenbeck, 1994) that only require base pairing of the very 3Ј-end bases between the primer and the template for strand elongation. In contrast, the promoter-dependent RdRps may require or favor the proximity of a promoter or promoter-like element in order to use primers efficiently. For example, both BMV and TCV RdRps were capable of primer elongation from their 3Ј-terminal negative strand initiation promoters, but they were incapable of primer elongation from primers hybridizing randomly at internal positions (Kao and Sun, 1996; . Also, the influenza virus and Thogoto virus RdRps can only initiate mRNA synthesis from their 3Ј-terminal promoters using dinucleotide or oligonucleotide primers, but not from internal sites (Seong and Brownlee, 1992; Leahy et al., 1997) . Base pairing between the primers and the template is not absolutely required in the Thogoto virus system, but influences the efficiency and site of initiation (Leahy et al., 1997) . Another example of promoter-dependent priming at the 3Ј end of plus strands has been proposed for poliovirus (Harris et al., 1994) . This model predicts that a protein cleavage product, termed Vpg, is uridylylated and then used to initiate minus strand synthesis by the poliovirus RdRp complex bound to the 3Ј-terminal promoter. In contrast to the multisubunit RdRps, the polymerase subunit of the poliovirus and the related hepatitis C virus RdRps are capable of nonsequence-specific primer extensions regardless of the presence of promoter sequences (Neufeld et al., 1991; Behrens et al., 1996) . The leader-primed RNA transcription in coronaviruses requires both short sequence complementarity between the primer and the template and the presence of a putative RdRp-binding sequence on the template RNA (Baker and Lai, 1990; Zhang and Lai, 1995) . In addition, the reverse transcriptase of the Mauriceville plasmid, which normally initiates DNA synthesis de novo at its 3Ј-terminal promoter, is capable of primer extension from its 3Ј-terminal promoter, even if the primer is not base paired with the template (Wang and Lambowitz, 1993) . Similarly to the above RdRps, the reverse transcriptase of the Mauriceville plasmid cannot efficiently use primers hybridizing randomly at internal positions (Chen and Lambowitz, 1997) . All these data suggest that primer-initiated strand elongation by a promoter-dependent RdRp or reverse transcriptase of the Mauriceville plasmid may be facilitated by a proximal promoter or promoterlike element. This is a novel phenomenon that may be characteristic of several promoter-dependent RdRps.
The finding that an RdRp-binding element facilitates in vitro 3Ј-TX and in vivo RNA recombination in TCV may have implications for other viral systems as well. For example, subgenomic RNA promoters or related sequences that bind their corresponding RdRp frequently are found as recombination sites in BMV (Allison et al., 1990) , Sindbis virus (Weiss and Schlesinger, 1991) and tobacco mosaic virus (Beck and Dawson, 1990) . Subgenomic RNA promoter-like elements may also have played a role in reshuffling functional 'modules' and in inter(intra-)viral recombination that facilitated viral evolution (Gibbs, 1987; Allison et al., 1989; Miller et al., 1995) . In addition, many crossover events in BMV (Nagy and Bujarski, 1992; Rao and Hall, 1993) , Sindbis virus (Hajjou et al., 1996) , tobraviruses (Goulden et al., 1991) , cucumoviruses (Fernandez-Cuartero et al., 1994) , alfalfa mosaic virus (Huisman et al., 1989) and barley stripe mosaic virus (Edwards et al., 1992) are located within 3Ј-or 5Ј-terminal promoter sequences. These promoter sequences, similarly to the subgenomic promoters, may have played a role in recombination events by recruiting the RdRp-nascent strand complex.
For the in vivo recombination studies, a derivative of the full-length, biologically active cDNA construct of sat-RNA C (pCAMD, Cascone et al., 1993) that contained ApaI and MluI sites at positions 206 and 142, respectively, combined with a 22 base, 5Ј-proximal deletion between positions 57 and 78 was mutagenized using the method of Kunkel (1985) with a partially degenerate primer (5Ј-CCCAGACCCtccagccaaa-GGGTAAATGGG-3Ј, where the lower case letters represent degenerate bases at a ratio of 95% wild-type to 1.66% each of the other three bases) to obtain G191C, U192A, G194A, G195U and U196C, respectively. Similarly, the pCAMD versions of UStem/3GC, LStem/3G, LStem/3GC, ∆U3 and C7 were generated with primers CXexgcL (5Ј-ATCCCA-GAGGGTCCAGCCAAACCCTAAATGGGC-3Ј, where the mutated bases are underlined), CX180-182 (5Ј-TCAAAAGAATGGGAGAC-CCT-3Ј), CX208-210 (5Ј-AAGGGTAAATCCCCCCAAAAACG-3Ј), CXdB (5Ј-CAGCCAAAGGGT∆∆∆TGGGCCCAA-3Ј, where the deleted bases are marked with ∆) and CX46 (5Ј-GACGCGTGAAAAC-ctggctgtttcccactcaaaaGAATCCCAGACC-3Ј), respectively. Mutants were identified by dideoxy sequencing using Sequenase (Amersham). The purified DNA representing each construct was linearized with EcoRI prior to Escherichia coli RNA polymerase transcription (Cascone et al., 1990) .
For the in vitro 3Ј-TX experiments, RNA templates were obtained by in vitro transcription with T7 RNA polymerase using either PCRamplified DNA templates or purified and linearized plasmid DNA (Song and Simon, 1994; . After phenol/chloroform extraction, unincorporated nucleotides were removed by repeated ammonium acetate/isopropanol precipitation (Song and Simon, 1994; . The obtained RNA transcripts were dissolved in sterile water and their amount and size were measured by a UV spectrophotometer and 5% polyacrylamide-8 M urea gel (denaturing PAGE) analysis (Song and Simon, 1994; .
CD-27 DNA was obtained with two sequential rounds of PCR, first using primers hairpin-1 [5Ј-GGG(A/T)(A/T)(A/T)(C/T)AGCCAGGG-AATTCGTGAAAACCTGGCTG-3Ј] and T7C3Ј (5Ј-GTAATACGACT-CACTATAGGGCAGGCCCCCCG-3Ј), and pT7C(ϩ) template (Song and Simon, 1994 ) that contains the full-length cDNA of sat-RNA C, followed by gel purification of the PCR product and a second round of PCR with primers har1/0 (5Ј-GGGAAACAGCCAGGGAATTCGTGA-3Ј) and T7C3Ј. CD-27mini DNA was also obtained by PCR with primers har1/0 and T7motif1 (5Ј-GTAATACGACTCACTATAGGGCTGCCG-CCGTTTTTGG-3Ј) using CD-27 as template. CD-13mini DNA was generated by PCR using primer pairs CD-13 (5Ј-GGACTCTTTCG-AGTGGGATACTGCCAGGGAATTCGTGA-3Ј) and T7motif1, while CD-14mini DNA was obtained with primer pairs SatD/hinc (5Ј-GTCG-ACTCTTTCGAGTGG-3Ј) and T7motif1 using CD-13mini as template, followed by cloning to the SmaI site of pUC19 and cleavage with HincII.
Constructs AU/GCmini, GC12mini and AU17mini were obtained by PCR with primers Har-AU (5Ј-GGGTTTAAATATTATATAGATATCTT-ATATAATATTTAACCCACTCAAAAGAATC-3Ј), Har-GC (5Ј-GGGC-GCGCGCGGTGATATCTCCGCGCGCGCCCACTCAAAAGAATC-3Ј) and AU-17 (5Ј-AAATATTATATTTAAATAGTATCTTATTTAATATA-ATATTTAACCCACT-3Ј), respectively, in combination with primer T7motif1 using CD-27 as template. Construct AU5mini was obtained by digesting AU17mini with DraI, followed by agarose gel purification of the cleaved product. Constructs CD-27mini/mot1, GC12mini/mot1 and AU/GCmini/mot1 were generated by PCR with primers T7motif1short (5Ј-GTAATACGACTCACTATAGGGCTGGAGGGTC-3Ј) and either Har1/0, Har-GC or Har-AU using CD-27, GC12mini and AU/ GCmini, respectively, as templates.
CD-14B was generated by cloning the PCR product obtained using pT7C(ϩ) DNA template with primers T7C3Ј and hairpin-satD [5Ј-GGG(A/T)(A/T)CCTTTCGAGTGGGATACTGCCAGGATATCGTAC-GGGAGCGTG-3Ј], into the SmaI site of pUC19. Two mutations were engineered into the priming stem of construct CD-14B that created a BamHI restriction site at the 3Ј end of the cDNA construct while maintaining the base pairing in the priming stem in the derived transcript. In addition, a unique EcoRV sequence was engineered into the loop region. The template DNA for T7 RNA polymerase transcription was linearized by BamHI cleavage. Derivatives of CD-14B with mutations in the motif1-hairpin or the priming stem, such as LStem/3G, LStem/ 3GC, U183G, U183G/A206C, U185G, U185G/A202C, ∆U3, UStem/ 3GC, A189U, G191C, U192A, C193U, G194A, G195U, U196C and C7, were generated by PCR with primers T7C3Ј and hairpin-satD using the corresponding pCAMD-derived templates described above and in Cascone et al. (1993) . The PCR-amplified DNA corresponding to each of the above constructs was cloned, linearized with BamHI and transcribed as described for CD-14B above. Sequence analysis of the obtained clones confirmed the presence of only the desired modifications.
Constructs Cmini and Cmini/mot1 were generated by PCR with the same 3Ј primer (Har1PCR, 5Ј-GAATTCCCTGGCTGTTTCCC-3Ј) and one of the 5Ј primers, T7motif1 or T7motif1-short, respectively, using CD-27 as template. Cmini/U183G was constructed with the method used for Cmini, except that U183G served as the PCR template.
Preparation of template-dependent RdRp from TCV-infected turnip plants, in vitro 3Ј-TX reactions (similar to transcription reactions, but using different RNA templates) and product analysis were carried out as previously described using a total of 20 µl of RdRp reaction mixture that contained 3 µg of template RNA. For the competition studies, 1 µg of CD-27 template RNA and increasing amounts of competitor RNA (as shown in Figure 4 ) were used in a 30 µl RdRp reaction mixture.
3Ј-TX products were treated with S1 nuclease or RNase H in the presence of 1 µg of loop1 oligo (5Ј-AATTCGTGAAAA-3Ј, complementary to the loop region of CD-27, see Figure 1C ) as described Simon, 1994, 1995) at 37°C for 1 h. After phenol/chloroform extraction and ammonium acetate/isopropanol precipitation, the products were analyzed on a 20 cm-long denaturing 5% polyacrylamide-8 M urea gel, followed by autoradiography and densitometry . The data were normalized based on the amount of template-directed radioactive UTP incorporated into the 3Ј-TX products and the molar amount of the template RNA in the RdRp reaction. For some experiments, the gels were stained with ethidium bromide, photographed and dried, followed by analysis with a phosphorimager as described .
The radioactively labeled 3Ј-TX product of CD-27 obtained from a 200 µl RdRp reaction was gel purified, then hybridized with loop1 oligo followed by digestion with RNase H as described above. The RNase H-cleaved product was gel purified and subjected to reverse transcription (RT) PCR with primer T7C3Ј and MMLV reverse transcriptase (Song and Simon, 1995) . Oligodeoxynucleotides T7C3Ј and Har1PCR (5Ј-GAATTCCCTGGCTGTTTCCC-3Ј) were used for PCR. The~210 base long PCR product was gel purified, cloned into the SmaI site of pUC19, and then sequenced using standard procedures (Sambrook et al., 1989) .
Turnip plants were inoculated with a mixture of sat-RNA C (CAMD series, see above), wild-type sat-RNA D and helper TCV RNA, as described previously (Cascone et al., 1993) . To test for the viability of sat-RNA C constructs, full-length sat-RNA C that contained mutations in the motif1-hairpin, but without the 22 nucleotide deletion (present in the CAMD series), were used for inoculation as descibed by Cascone et al. (1993) . Total RNA isolation, Northern blot and RT-PCR analysis of sat-RNA C progeny were done according to a published procedure (Cascone et al., 1993) .