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Department of Plant Pathology, University of Kentucky, Lexington, Kentucky
Received 29 July 2003/ Accepted 31 October 2003
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The most popular model of RNA recombination is the template switching (copy choice) mechanism, which suggests that the viral RNA-dependent RNA polymerase (RdRp) switches templates during cRNA synthesis (8, 10, 20). After the jump from the donor to the acceptor RNA, the RdRp resumes RNA synthesis by using the nascent RNA (which has been made on the donor RNA) as a primer. On the basis of the role of base pairing between the acceptor RNA and the primer (nascent RNA), RNA recombination events are divided into three categories: base pairing dependent (similarity essential), base pairing assisted, and base pairing independent (similarity nonessential) (20). RNA recombination is probably a chance event; thus, each nucleotide in an RNA molecule may serve as a target for recombination. Most experimental data demonstrate that not all regions within an RNA are equally recombinogenic. The sequences that participate in RNA recombination at higher and lower frequencies are called hot and cold spots, respectively. Various models have been proposed to explain the occurrence of the observed hot and cold spots for different viruses (8, 10, 20).
Tombusviruses are single-component plus-stranded RNA viruses of plants, and they are known to support RNA recombination at high frequency (2, 30-33). Although the involvement of the genomic RNAs (gRNAs) in recombination is well documented for tombusviruses, DI RNAs associated with these viruses are the most popular templates in studies of RNA recombination. This is because (i) they are involved in RNA recombination with high frequencies (2, 30-33), (ii) they do not contribute essential protein factors to replication, and thus (iii) they have greater genetic plasticity than the viral gRNA. Importantly, recombination in DI RNAs is thought to occur by using the same mechanism as recombination involving the viral gRNA (30). The replication process of tombusviruses and the associated DI RNAs is carried out by the replicase complex, which includes two viral proteins and unknown host factors (17, 22, 28). The tombusvirus DI RNAs, such as Tomato bushy stunt virus (TBSV)-associated DI-73 (Fig. 1A), contain three or four noncontiguous genomic segments (30). The two or three sequence deletions leading to DI RNA formation are thought to be the consequence of viral replicase jumping on the genomic template, and the deletions may occur in a stepwise manner (30).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All constructs that contained AU, GC2, R', or AUs sequences (Fig. 1A) were obtained by inserting the PCR-amplified (by using the primers listed in Table 1) AU sequence (including a segment from the 3' end of BMV RNA3, between positions 197 and 242) (12), GC2 (11), R' (13), or the artificial AUs (12) segments into DI-FP either at the 5' side of the FP sequence (at the unique XbaI and NcoI restriction sites) or at the 3' side (at the unique MluI and BamHI restriction sites). The RII deletion constructs (derivatives of DI-AU1-FP-AU2) were obtained by amplifying the RII and RIII/IV sequences of DI-FP with the primer sets shown in Table 1, followed by treatment with BamHI and SalI, and cloning to the similarly treated DI-AU1-FP-AU2 or DI-FP (Fig. 1A) construct. All of the clones were sequenced to confirm the desired changes.
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Preparation of CNV gRNA and DI RNA transcripts in vitro. To generate the gRNA of Cucumber necrosis virus (CNV) and the DI RNA transcripts, we linearized pK2/M5p20STOP (27) and all of the DI RNA clones, respectively, with SmaI, followed by in vitro transcription with T7 RNA polymerase (18, 19). In vitro-generated transcripts of DI RNAs were purified from 1% agarose gel, phenol-chloroform extracted, precipitated in 95% ethanol, and washed three times with 70% ethanol to remove residual salts. The in vitro RNA transcripts were analyzed in 1% agarose gels and quantified with a UV spectrophotometer (Beckman).
For the in vitro RdRp experiments, RNA templates were obtained by in vitro transcription reaction with T7 RNA polymerase (see above and references 4 and 17). The templates and primers used for PCR are listed in Table 1. The unincorporated nucleotides were removed by phenol-chloroform extraction and repeated ammonium acetate-isopropanol precipitation (4, 17). The T7 transcription products were analyzed by 5% denaturing polyacrylamide gel electrophoresis (PAGE), and the amounts of RNA were measured with a UV spectrophotometer.
Preparation and electroporation of protoplasts. Nicotiana benthamiana protoplasts were prepared as described before (24). Briefly, N. benthamiana callus was treated with 0.5 g of cellulysin and 0.1 g of macerase (Calbiochem) for 4.5 h in protoplast incubation medium (18) at 25°C and then washed twice with 0.5 M mannitol and once with electroporation buffer (10 mM HEPES, 10 mM NaCl, 120 mM KCl, 4 mM CaCl2, 200 mM mannitol). For electroporation, we used 5 x 105 protoplasts, 5 µg of CNV gRNA, and 1 µg of DI RNA. Electroporation was performed with a Gene Pulser II (Bio-Rad) at a voltage of 0.2 kV and a capacitance of 0.5 µF. After electroporation, the samples were left on ice for 30 min and then 1.8 ml of protoplast culture medium was added (18). Protoplasts were incubated in petri dishes (35 by 10 mm) in the dark for 24 to 48 h at 22°C.
N. benthamiana plants were inoculated with 3 µg of CNV gRNA and 1 µg of DI RNA (prepared as described above) by rub inoculation as described earlier (17). Plants were incubated for 10 days in a temperature-controlled (
22°C) room.
Total RNA extraction from protoplasts and plants and RNA analysis.
Total RNA was extracted from protoplast by the phenol-chloroform method (18). Aliquots of total RNA were analyzed on 1.2% agarose gels. RNA samples were treated with formamide at 85°C before being loaded onto the gel. For Northern blot analysis, RNA was transferred to Hybond XL membrane (Amersham-Pharmacia) by electrotransfer and hybridized with DI-72-specific probes [RI(-)] (24). Hybridization with 32P-labeled RNA probes was performed in ULTRAhyb hybridization buffer at 68°C under the recommended conditions (Ambion). The 32P-labeled RNA probes were made in an in vitro transcription reaction with T7 RNA polymerase in the presence of [
-32P]UTP and a PCR template obtained with primers #15 (5'-GTAATACGACTCACTATAGGGCATGTCGCTTGTTTGTTG-3') and #20 (5'-GGAAATTCTCCAGGATTTCTC-3') (24).
RT-PCR analysis, cloning, and sequencing of recombinant DI RNAs. To obtain recombinant DI RNA clones, a reverse transcriptase (RT) reaction was done with primer #106 (5'-ACCTGGAAGCTTATGCCAGATTTACACTCATC-3') and 2 µl of total RNA (16). This was followed by amplification by PCR with primers #106 and #380 (5'-GGACGAATTCCATAATTATTATCTTTAFTTG-3').
The RT-PCR products were digested with EcoRI and HindIII, followed by gel isolation and ligation into a similarly treated pUC19 vector. The clones for sequencing were selected after restriction digestion with EcoRI and HindIII. The sequencing was done with a CEQ Cycle Sequencing Kit (Beckman) and primer #553 (5'-GTAAAACGACGGCCAGT-3').
In vitro RNA binding studies.
The RNA probe in the gel shift experiments was RIII(-)/cPR11 (23), containing the 82-nucleotide (nt) minus-stranded RIII replication enhancer and the 11-nt minimal promoter for plus-strand synthesis (23). The labeling was done with [-32P]UTP and T7 RNA polymerase (26). Competitor RNAs (see Fig. 6) were prepared with T7 polymerase on PCR-amplified templates with primers described in Table 1. The gel shift experiments were performed as described by Rajendran and Nagy (26a). Briefly, various amounts of competitor RNAs (used in 5x, 15x, and 45x excesses over the constant amount of the [-32P]UTP-labeled RNA probe) were mixed with 1 µM recombinant p33 and p92 preparations in the presence of 50 mM Tris-HCl (pH 8.2), 10 mM MgCl2, 10 mM dithiothreitol, 10% glycerol, 2.4 U of RNase inhibitor, and 100 ng of tRNA. After 10 min of preincubation at 25°C, 2 ng of [-32P]UTP-labeled RNA probe was added to each RNA-binding reaction mixture, which was further incubated for 25 min. The samples were then analyzed by electrophoresis on native 4% polyacrylamide gels run at 200 V for 60 min at 4°C in Tris-glycine buffer (25 mM Tris, 190 mM glycine, 5 mM EDTA, pH 8.5). Dried gels were analyzed with a PhosphorImager. The recombinant proteins were purified from Escherichia coli as described earlier (24, 26).
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1 µg of RNA transcripts (the amounts of RNA templates were adjusted on the basis of their sizes to have similar molar amounts of templates in each reaction mixture). RdRp reactions were carried out as previously described (17). The reaction was terminated by adding 5 µl of 10% sodium dodecyl sulfate, followed by phenol-chloroform extraction and ammonium acetate-isopropanol precipitation (17). The RdRp products were analyzed by 5% denaturing PAGE in the presence of 8 M urea, followed by PhosphorImager analysis. The data for each sample were normalized on the basis of the amount of templated UTP incorporated (4, 17). |
RESULTS |
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Previous works defined BMV-derived and artificial AU-rich sequences that served as recombination-promoting signals (Fig. 1) during BMV replication in vivo (12-14, 16). Frequent generation of recombinants was only observed when the two recombining RNA templates carried the same or similar AU-rich sequences. Because recombination sites in BMV were located within the AU-rich regions, it was proposed that the BMV replicase recognized the AU-rich signals during the recombination events (12-14, 16). We wanted to test if these AU-rich sequences could also promote RNA recombination in tombusviruses.
The control TBSV DI RNA-based recombination vector does not support RNA recombination. To study RNA recombination in tombusviruses, first we developed a recombination vector, which facilitated the construction and testing of additional parental DI RNAs (Fig. 1A). The recombination vector, named DI-FP, is based on TBSV DI-73 RNA carrying a 117-nt insert of nonviral origin (derived from dEGFP [BD Biosciences]) between regions I and II (RI and RII; Fig. 1A). To avoid possible artifactual RNAs (i.e., unwanted recombinant-like RNAs) made during plasmid propagation in E. coli and/or during RNA transcription with T7 polymerase, we gel isolated DI-FP RNA transcripts prior to their use. In addition, to exclude those DI RNAs that might be generated spontaneously from the helper gRNA during the infection, a process known as de novo DI RNA formation (27, 30), we used the heterologous virus CNV (closely related to TBSV) as the helper virus. CNV can efficiently support the replication of TBSV DI-73 RNA and the DI-FP vector (24) (Fig. 1C).
The gel-isolated DI-FP transcripts were coelectroporated with CNV gRNA transcripts into N. benthamiana protoplasts as described earlier (24). We followed the accumulation of DI-FP RNA in protoplasts (termed zero passage; Fig. 1C) by Northern blotting with a DI RNA-specific probe [RI(-); Fig. 1A] and RT-PCR (primers #160 and #380; Fig. 1A). These experiments detected only the parental DI-FP RNAs in the zero-passage protoplast samples, suggesting a lack of recombinant accumulation (Fig. 1C). The total RNA obtained after 48 h of incubation in zero-passage protoplasts was used for electroporation into a new batch of protoplasts (termed first passage). Northern blot and RT-PCR analyses revealed that the DI-FP RNA recombination vector replicated efficiently and stably in the first-passage protoplasts (Fig. 1D to F, lanes 1 to 4). Note that we made sequential passages of the progeny DI RNAs from one batch of protoplasts to another, since there is no cell-to-cell spread of tombusviruses in protoplasts. Altogether, we performed three sequential passages, yet the DI-FP vector did not appear to support recombinant DI RNA accumulation (data not shown).
An AU-rich sequence supports RNA recombination efficiently in TBSV-associated DI RNA. To test the effect of an AU-rich sequence on tombusvirus recombination, we inserted the 69-nt-long AU sequence (Fig. 1B; this sequence is identical to the AU1 sequence used in the study described in reference 12), which had been tested previously for recombination in BMV, into DI-FP in such a way that identical copies of the AU sequence flanked the dEGFP-derived FP region at the 5' and 3' sides, respectively (termed AU1 and AU2 in the construct DI-AU1-FP-AU2; Fig. 1A), resulting in a repeated sequence. It was predicted, on the basis of the BMV results (12), that recombination might take place between the repeated copies of the AU sequence, thus resulting in deletion of one of the AU copies plus the FP sequence (Fig. 1A) located between the repeated AU sequences.
Incubation of the protoplast cells after coelectroporation of the gel-purified DI-AU1-FP-AU2 RNA with the CNV gRNA resulted in efficient amplification of the parental-size DI RNA, suggesting that the insertions did not debilitate DI RNA replication (Fig. 1C). No recombinants were detected in these protoplasts by Northern blotting, while RT-PCR analysis did show the occurrence of novel RNAs that were shorter than the input RNAs by 100 to 250 bp (not shown). This indicated that the putative recombinant DI RNAs were present in the zero-passage protoplasts at low levels that could only be detected by the more sensitive RT-PCR assay. However, a passage of the total RNA to a new batch of protoplasts, followed by incubation, resulted in recombinant-like DI RNAs in 100% (15 out of 15) of the experiments on the basis of total RNA (Fig. 1D, lanes 6 to 9), Northern blot (Fig. 1E), and RT-PCR (Fig. 1F) analyses. Interestingly, the sizes of the novel DI RNA recombinants were variable, suggesting that the recombination events were "imprecise" in nature. Indeed, cloning and sequencing of these putative recombinants confirmed that (i) they derived from DI-AU1-FP-AU2 RNA via deletions and (ii) the recombination junction sites were different in many of the recombinants (Fig. 2A). All of the 5' deletion sites in the 15 recombinants sequenced were located within the AU sequence (i.e., AU1), while the 3' deletion sites were clustered mostly within RII, which flanks the inserted sequences in DI-AU1-FP-AU2 (Fig. 2A). Overall, we did not find the generation of precise (homologous) recombinants between the duplicated AU sequences, which were the most common recombinants in the BMV system (12).
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The isolated DI RNA recombinants from protoplasts appear to be true recombinants, since they can be detected by gel analysis of the total RNA (Fig. 1D) and by Northern blotting (Fig. 1E). In addition, control RT-PCR assays performed on gel-isolated DI RNA transcripts (the same DI RNA transcripts that were used for electroporation to protoplasts) did not detect recombinant-sized DI RNAs for these constructs (Fig. 1F, lanes 5, 10, and 15).
To test if the length of the inserts is an important factor in recombination events, we generated a DI RNA carrying an 152-nt-long sequence from the barstar gene (Fig. 1A) (7). The resulting DI-152Bar* RNA was stable in protoplasts after one (Fig. 1D to F, lanes 16 to 19) or two (data not shown) passages. This result, together with that of DI-FP, supports the notion that DI RNAs with short inserts can be stable in protoplasts (under the conditions used) if they lack recombination-promoting signals.
To determine if recombination might occur in whole plants as well, we tested the accumulation of DI-FP, DI-AU1-FP-AU2, and DI-AU1-FP RNAs (in the presence of the CNV gRNA) in N. benthamiana plants 10 days after inoculation with gel-purified transcripts. Northern blot (Fig. 1G) and RT-PCR (data not shown) analyses demonstrated that the systemically infected (uninoculated) leaves contained the parental-size DI RNAs for each DI RNA tested. In contrast, recombinant-like DI RNAs appeared only in DI-AU1-FP-AU2- and DI-AU1-FP RNA-containing plants (Fig. 1G, lanes 5 to 12) and not in DI-FP-containing plants (Fig. 1G, lanes 1 to 4). Cloning and sequencing of a representative number of recombinants confirmed that the accumulating recombinants in plants are similar to those observed in protoplasts, with most of the junction sites located within the 5' AU1 sequence and RII (Fig. 2B and D).
RII sequence affects the distribution of recombination sites.
Since many recombinants generated with DI-AU1-FP-AU2 and DI-AU1-FP RNAs had the recombination sites within a 40-nt stretch close to the 5' end of RII(+) (we term this region the hs40 hot spot), it is possible that sequences around hs40 might influence the selection of recombination sites. To test if hs40 is required for replication of DI RNA, we generated a series of DI constructs based on various 5' deletions in RII. First, we tested construct DI-FP-RII/
40 (Fig. 3A), which has the same sequence as the DI-FP vector (Fig. 1A) but lacks the 5' 40 nt from RII (i.e., hs40). DI-FP-RII/40 RNA accumulated efficiently in protoplasts, and no recombinants were detected in protoplasts after the first passage (Fig. 3B and C, lanes 1 to 5). This suggests that the hs40 sequence is not essential for DI RNA accumulation in protoplasts.
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20, which lacked 20 nt from the 5' end of RII(+) in DI-AU1-FP-AU2, generated recombinants as efficiently as DI-AU1-FP-AU2 did after the first passage (Fig. 3B and C, lanes 7 to 11). Interestingly, the distributions of recombination sites in the recombinants obtained with DI-AU1-FP-AU2 (Fig. 2A) and DI-RII/20 (Fig. 4A) were similar, except that 50% (7 out of 15) of the recombinants obtained with DI-RII/20 contained precise recombination sites between the duplicated AU sequences. The second construct tested, DI-RII/40, which lacked 40 nt from the 5' end of RII(+) in DI-AU1-FP-AU2, also generated recombinants in 100% of the samples (Fig. 3B and C, lanes 13 to 17). Interestingly, 87% (26 of 30) of the 3' junctions were within the AU2 copy and only 13% were within the RII sequence. We also observed precise recombinants between the repeated AU regions in 36% (11 of 30) of the recombinants (Fig. 4B). The occurrence of precise recombinants may suggest that primer realignment (base pairing between the primer and the acceptor region) assisted by the presence of duplicated sequences may take place during these recombination events (see Discussion).
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60, which lacked 60 nt from the 5' end of RII(+) in DI-AU1-FP-AU2, supported recombination as efficiently as DI-AU1-FP-AU2 (Fig. 3B and C, lanes 19 to 23). Cloning and sequencing revealed that all 15 recombinants, each derived from separate samples, were precise recombinants (Fig. 4C) with junctions between the duplicated AU sequences. In contrast to the above DI RNA constructs, DI-RII/100, which lacked 100 nt from the 5' end of RII(+), did not support the generation of recombinants in protoplasts (Fig. 3B and C, lanes 25 to 29). Moreover, DI-RII/100 accumulated only inefficiently (tested after the first passage) in protoplasts, suggesting that an important cis-acting signal may have been deleted from this construct (see Discussion). Overall, these results suggest that the RII sequence may have two important effects on RNA recombination: first, it could affect the frequency of recombinant formation, and second, it could also influence the selection of recombination sites. In addition, we conclude that hs40 is not required for recombination to take place within the RII sequence.
Short, AU-rich sequences can also support RNA recombination in DI RNA.
To test if shorter, AU-rich sequences could also support RNA recombination in tombusviruses, first we deleted the BMV-derived R' region (40 nt long; Fig. 1B) from the 69-nt AU1 sequence in DI-RII/40. The resulting construct, DI-AUs-FP-AU2, contained only the 29-nt-long artificial AU-rich sequence (termed AUs, with 76% AU content), yet it still supported recombination efficiently with most of the junction sites located within the 5' AUs and 3' AU2 regions (Fig. 5A). Note that we found that the distribution of the recombination sites was similar to that observed for DI-RII/40 recombinants, with almost 50% (7 of 17) of the recombinants having precise junctions between the repeated AU-rich sequences (Fig. 5A).
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75% AU content; Fig. 1B), it was still active in recombination (Fig. 5B). Most of the recombinants obtained with DI-R'-FP-AU2 (Fig. 5B) contained precise junctions between the repeated AU-rich sequences. Overall, this suggests that both the BMV-derived R' and the artificial AUs portions of the original AU sequence are capable of supporting RNA recombination efficiently in TBSV. We conclude that an AU-rich sequence as short as 21 to 29 nt can promote recombination in TBSV DI RNA. GC-rich sequences cannot "silence" RNA recombination in TBSV. Since RNA recombination promoted by the AU sequence could efficiently be inhibited by the presence of GC-rich sequences located 3' of the AU sequence (referred to here in the plus-strand orientation) in the BMV RNAs (11), we also tested the effect of one of these well-characterized sequences (termed GC2, with 55% GC content) (11) on DI RNA recombination. First, the GC2 sequence was inserted 3' of the R' sequence in DI-R'-FP-AU2 (Fig. 5B). The resulting construct, DI-R'/GC2-FP-AU2 (Fig. 5C), supported RNA recombination efficiently. Most of the junctions were located within the R' sequence and the 3' AU2 sequence, including more than 60% (9 of 15) with precise junctions between the repeated AU-rich sequences (Fig. 5C). Second, placing the GC2 sequence behind the R' sequence within the 3' repeat (construct DI-AU1-FP-R'/GC2) did not inhibit recombination that took place between the 5' AU1 sequence and either R', GC2, or the RII sequences (Fig. 5C). Interestingly, none of the isolated recombinants contained precise junctions between the repeated AU-rich sequences, suggesting that the GC2 sequence interfered with precise recombination but not with imprecise recombination.
Minus-stranded RII binds efficiently to the TBSV RdRp. Previous in vitro studies with the CNV RdRp (4) predicted that the same AU sequence tested as described above is a good donor, while it is a poor acceptor during template switching events. On the basis of these observations, it is possible that RNA recombination might occur during plus-strand synthesis when the RdRp would "jump" from the AU1(-) region to the RII(-) region (around hs40) (see Discussion). Thus, this model predicts that RII(-) should contain a binding site(s) for the tombusvirus replicase proteins. In contrast, the heterologous dEGFP-derived FP sequence is expected to lack a high-affinity binding site(s), thus serving as a cold spot during the recombination events. This model was tested as described below in two different in vitro assays.
First, we have tested the ability of the purified recombinant TBSV replicase proteins (i.e., p33 and p92 expressed and purified from E. coli) (26a) to bind to RII(-) sequences or the FP sequence (also tested in the complementary orientation). To obtain quantitative results, we used template competition in a gel mobility shift assay (26a). The experiments included the same amount of 32P-labeled template [derived from the RIII(-) replication enhancer] (23) and the same amount of purified recombinant p92 (Fig. 6B) or p33 (Fig. 6C) protein. The amounts of unlabeled competitors were used in 5x, 15x, and 45x excesses over the labeled RNA for each competitor RNA. These experiments revealed that the two competitor RNAs that contained overlapping portions of the RII(-) sequence (namely, construct #7 and #18, Fig. 6A) were two- to threefold better competitors for binding to p92 or p33 (Fig. 6B and C, lanes 4 to 9) than the FP control sequence (lanes 1 to 3). Therefore, this supports the model in which RII(-) is a hot spot region because of its increased binding to the replicase proteins (see Discussion).
Since binding to a template does not necessarily result in productive interaction between the particular sequence and the RdRp, we also tested the ability of the partially purified CNV RdRp to use templates containing various portions of the RII(-) sequence and of the FP(-) sequence in the presence of a short primer in an in vitro primer extension assay (Fig. 7) (4). As shown earlier, primer extension by the CNV RdRp in the in vitro assay depends on (i) the ability of the template region to interact with the CNV RdRp and (ii) the number of base pairs formed between the primer and the template (4). Since all of the constructs tested in this work contained the same 5-bp primer-template region (termed Art-5, which supported self-priming of RNA synthesis on the template by the CNV RdRp; see reference 4), we predicted that the activity of a particular template in the CNV RdRp reaction should depend on the ability of the template region to interact with the CNV RdRp. These experiments revealed that primer extension was about three- to ninefold more efficient when it started within the RII(-) sequence than the primer extension from the FP sequence (compare constructs 16, 17, and 18 with constructs 20 and 21 in Fig. 7A to C), while primer extension from the 3' end of RII(-) was almost twofold more efficient (construct 11). Interestingly, the most efficient constructs were those that contained the primer within the hs40 hot spot region (see constructs 16 and 17) or in the middle of RII(-) (construct 17, Fig. 7), suggesting that this region contains a putative cis-acting element (see Discussion). Overall, data from the primer extension experiments do support the model in which RII(-) might be active in recombination owing to its enhanced binding to the RdRp.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Indeed, we found that an AU-rich sequence that promoted RNA recombination in BMV also facilitated RNA recombination in a TBSV-associated DI RNA. The supporting evidence includes the following: (i) the recombination sites were frequently located within the AU-rich region, and (ii) the recombination frequency of the DI RNAs containing AU-rich sequences was higher than that of the control DI RNAs carrying dEGFP-derived or barstar sequences (Fig. 1). We also obtained data that support a different role for the AU-rich sequence in tombusvirus recombination than in the case of BMV. These include the facts that (i) only one copy of the AU-rich sequence was enough for promoting tombusvirus recombination, while two copies were needed for efficient recombination in BMV; (ii) most of the recombination events occurred precisely or semiprecisely between the two copies of AU-rich sequences in BMV, while this type of recombinants was less frequent in the case of TBSV DI RNA; and (iii) an additional viral sequence (i.e., RII) was also involved in RNA recombination in TBSV DI RNA, while it is unknown if sequences other than the AU-rich regions are involved in facilitating the occurrence of recombination between the AU-rich sequences in BMV. Overall, the above similarities and differences between the two recombination systems suggest that the AU-rich sequences can promote recombination in both viruses, but the selection of recombination sites (which are probably determined during resolution of the putative recombination intermediates) is different for the majority of recombinants (mostly precise for BMV and largely imprecise for TBSV recombinants). This observation is also supported by the different effects of GC-rich sequences on recombination in the two viral systems. While GC-rich sequences located downstream on the acceptor template (on the basis of the progress of the viral replicase on the template) inhibited or silenced recombination in BMV (11), we did not observe similar effects of the same GC-rich sequence in the case of TBSV DI RNA (Fig. 5C and D).
In addition to the in vivo data discussed above, in vitro data obtained with the purified BMV (9) and CNV (4, 4a) replicases also support the role of AU-rich sequences when present in the donor RNA. For example, the end-to-end template switching assay developed by Kim and Kao (9) demonstrated that the AU-rich stretches when present at the 5' end of the donor RNA, where the nascent strand must be released from the donor RNA before template switching occurs, facilitated end-to-end recombination events. Interestingly, the AU-rich sequence tested in this work also supported template switching by the partially purified CNV RdRp in vitro (4, 4a). The template RNA containing the AU-rich sequence served primarily as a donor RNA in the in vitro CNV RdRp assay (4, 4a), giving valuable insight into the significance of sequence context in RNA recombination in vitro, and possibly in vivo.
Further evidence of the possibility that AU-rich sequences could serve as common recombination signals comes from studies with retroviruses. With an in vitro template switching assay based on purified RT from Human immunodeficiency virus, it has been demonstrated that AU-rich sequences promoted RNA recombination (5, 36). Since the RT favors base pairing between the nascent strand and the acceptor strand prior to resumption of cDNA synthesis, most of the in vitro recombinants were precise (i.e., occurred precisely within homologous regions), although recombinants with extra nucleotides, mismatched nucleotides, or short deleted regions at the recombination sites were also isolated (5, 36). The observation that AU-rich sequences form hot spots in recombinants obtained with BMV, retrovirus, and TBSV suggests that the induction of recombination by AU-rich sequences is similar for these viruses. Moreover, the resolution of recombination intermediates may use somewhat similar mechanisms for BMV and retroviruses, but it is different in tombusviruses. We propose that the difference is due to two factors. (i) The BMV RdRp and the retrovirus RT favor a somewhat precise annealing step between the nascent strand (primer) and the acceptor strand prior to the resumption of RNA-DNA synthesis, while the tombusvirus RdRp does not seem to favor this step. Indeed, we have proposed that the CNV RdRp can easily resume RNA synthesis (primer extension) without the need for extensive base pairing between the primer and the acceptor strands (4). (ii) There might be differences among these viruses in the use of cis-acting elements to guide the jumping viral replicase to a new acceptor site before resumption of primer extension, resulting in recombination hot spots (see below). It is also important to note that we cannot completely rule out the possibility that selection for the best-fit recombinants also affects the types of recombinants isolated in these virus infections. In summary, in vitro and in vivo data suggest that AU-rich sequences might serve as common recombination signals. However, the likely differences in recombination between viruses are caused by (i) differences in template sequence recognition during the template switching event and (ii) various roles for base pairing between the primer and template during the template switching events.
Model of AU-rich sequence-driven recombination in tombusviruses. Since both plus- and minus-strand synthesis takes place during DI RNA replication in protoplasts, it is difficult to establish whether plus- or minus-stranded RNAs are used as templates for recombination. However, on the basis of previous in vitro experiments with the partially purified CNV RdRp, the likely role of the AU-rich sequence is to promote recombination at the donor sites (4a). Moreover, the same AU sequence used in this study was found to be a relatively poor template (in comparison with known cis-acting elements of tombusviruses) in in vitro primer extension and template switching experiments with the CNV RdRp (4, 4a), suggesting that the AU sequence is unlikely to form a recombination hot spot as an acceptor site. If this is the case in protoplasts as well, then most of the recombination events might involve minus-stranded templates and thus, template switching would occur during plus-strand synthesis (Fig. 8A). This is because the first copy of the repeated AU regions (i.e., AU1), which is observed as a hot spot with DI-AU1-FP-AU2 (Fig. 2A), would be at the 3'-proximal location (relative to the deletion junctions) in the minus-stranded DI-AU1-FP-AU2 RNA (Fig. 8A). Thus, the AU1 repeat is favorably positioned to serve as a donor site for promoting jumping events by the tombusvirus replicase to a new location [which is hs40 within RII(-); see below]. The above model (Fig. 8A1 and A2) also predicts that the second AU repeat (i.e., AU2), located at a more 5' position in the minus-stranded DI-AU1-FP-AU2 RNA, should be less favorable as a donor site, because this region could only be copied by the RdRp after the first AU1 repeat had already been copied. Accordingly, the lack of an AU2 repeat in DI-AU1-FP RNA did not significantly alter the recombination sites or the frequency of the recombination events (Fig. 1 and 2). Interestingly, the AU sequences have also been predicted to support recombination during plus-strand synthesis when they were present in the minus-stranded BMV RNAs (12).
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40 and DI-RII/60; Fig. 4). To explain the formation of these precise recombinants, we propose that the RdRp might still bind to the RII(-) region during the jumping event, but annealing (base pairing) between the primer and the template might take place within the AU2 repeat. This annealing step might then promote primer extension initiation from the AU2 repeat, therefore resulting in precise or semiprecise recombination (Fig. 8B1 and B2). We propose that a similar annealing step between the primer and the acceptor region might be inhibited in the presence of extra sequences between AU2 and RII(-) (see construct DI-AU1-FP-AU2, Fig. 1), thus favoring the occurrence of recombination within the hs40 region that flanks the putative cis element in RII(-) (Fig. 8A1 and A2). A surprising observation in this work is the discovery of the major role of RII in TBSV recombination. The in vitro binding studies with the recombinant TBSV p33 and p92 replicase proteins and the primer extension studies with the CNV RdRp preparation suggest that there is a putative cis-acting element in RII(-) located 5' of the hs40 sequence. Indeed, construct 18, which contained the 5' half of RII(-) (between positions 100 and 239, Fig. 6 and 7), competed efficiently for binding to p33/p92 (Fig. 6) and was an efficient template for primer extension in the in vitro CNV RdRp assay (Fig. 7). We propose (Fig. 8A2) that the jumping viral replicase is likely guided by the RII(-) cis-acting element before resumption of primer extension, resulting in recombination hot spots. The role of this cis element is likely more important than the effect of base pairing between the primer and the template during tombusvirus recombination events. Accordingly, we observed earlier that the CNV RdRp could perform RNA synthesis (primer extension and template switching) without the need for extensive base pairing between the nascent strand and the acceptor strand (4, 4a). This observation can explain why there are not long stretches of sequence identity around the recombination sites generated in infections with DI-AU1-FP-AU2 (Fig. 2A).
The role of cis-acting replication elements in recombination has been proposed before in several viral systems (reviewed in reference 20). For example, a known replication enhancer element (19) in a satellite RNA (termed satC), which is associated with Turnip crinkle virus infections, promoted recombination between mutated satC and satD, another satellite RNA (3, 21). It has been proposed that the replication enhancer of satC is involved in binding to the jumping Turnip crinkle virus replicase (18, 19), a process similar to the role that was proposed above for RII(-) in TBSV DI RNA (Fig. 8). A different class of cis-acting element, namely, the subgenomic promoter region, has also been proposed to promote RNA recombination in luteoviruses (10a) and BMV (34). In summary, the discovery of the role of the RII(-) element in RNA recombination, in combination with published data cited above, suggests that cis-acting elements might play much wider roles in viral RNA recombination than previously anticipated.
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ACKNOWLEDGMENTS |
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This work was supported by NIH, the Kentucky Science and Engineering Foundation, the Kentucky Tobacco Research and Development Center at the University of Kentucky, and a USDA special grant (for sequencing).
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FOOTNOTES |
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Publication 03-12-093 of the Kentucky Agricultural Experiment Station.
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