Mechanism of RNA recombination in carmo- and tombusviruses: evidence for template switching by the RNA-dependent RNA polymerase in vitro

RNA recombination occurs frequently during replication of tombusviruses and carmoviruses, which are related small plus-sense RNA viruses of plants. The most common recombinants generated by these viruses are either defective interfering (DI) RNAs or chimeric satellite RNAs, which are thought to be generated by template switching of the viral RNA-dependent RNA polymerase (RdRp) during the viral replication process. To test if RNA recombination is mediated by the viral RdRp, we used either a purified recombinant RdRp of Turnip crinkle carmovirus or a partially purified RdRp preparation of Cucumber necrosis tombusvirus. We demonstrated that these RdRp preparations generated RNA recombinants in vitro. The RdRp-driven template switching events occurred between either identical templates or two different RNA templates. The template containing a replication enhancer recombined more efficiently than templates containing artificial sequences. We also observed that AU-rich sequences promote recombination more efficiently than GC-rich sequences. Cloning and sequencing of the generated recombinants revealed that the junction sites were located frequently at the ends of the templates (end-to-end template switching). We also found several recombinants that were generated by template switching involving internal positions in the RNA templates. In contrast, RNA ligation-based RNA recombination was not detected in vitro. Demonstration of the ability of carmo- and tombusvirus RdRps to switch RNA templates in vitro supports the copy-choice models of RNA recombination and DI RNA formation for these viruses.     

Engineering of homologous recombination hotspots with AU-rich sequences in brome mosaic virus.

Previously, we observed that crossovers sites of RNA recombinants clustered within or close to AU-rich regions during genetic recombination in brome mosaic bromovirus (BMV) (P. D. Nagy and J. J. Bujarski. J. Virol. 70:415-426, 1996). To test whether AU-rich sequences can facilitate homologous recombination, AU-rich sequences were introduced into parental BMV RNAs (RNA2 and RNA3). These insertions created a homologous RNA2-RNA3 recombination hotspot. Two other AU-rich sequences also supported high-frequency homologous recombination if a common sequence with high or average G/C content was present immediately upstream of the AU-rich element. Homologous RNA recombination did not require any additional sequence motifs or RNA structures and was position nonspecific within the 3' noncoding region. These results suggest that nucleotide content (i.e., the presence of common 5' GC-rich or moderately AU-rich and 3' AU-rich regions) is the important factor that determines the sites of homologous recombination. A mechanism that involves replicase switching during synthesis of positive-sense RNA strands is presented to explain the observed results.     

Authentic replication and recombination of Tomato bushy stunt virus RNA in a cell-free extract from yeast

To study the replication of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we have developed a cell-free system based on a Saccharomyces cerevisiae extract. The cell-free system was capable of performing a complete replication cycle on added plus-stranded TBSV replicon RNA (repRNA) that led to the production of approximately 30-fold-more plus-stranded progeny RNAs than the minus-stranded replication intermediate. The cell-free system also replicated the full-length TBSV genomic RNA, which resulted in production of subgenomic RNAs as well. The cell-free system showed high template specificity, since a mutated repRNA, minus-stranded repRNA, or a heterologous viral RNA could not be used as templates by the tombusvirus replicase. Similar to the in vivo situation, replication of the TBSV replicon RNA took place in a membraneous fraction, in which the viral replicase-RNA complex was RNase and protease resistant but sensitive to detergents. In addition to faithfully replicating the TBSV replicon RNA, the cell-free system was also capable of generating TBSV RNA recombinants with high efficiency. Altogether, tombusvirus replicase in the cell-free system showed features remarkably similar to those of the in vivo replicase, including carrying out a complete cycle of replication, high template specificity, and the ability to recombine efficiently.     

Frequent homologous recombination events between molecules of one RNA component in a multipartite RNA virus

Brome mosaic bromovirus (BMV), a tripartite plus-sense RNA virus, has been used as a model system to study homologous RNA recombination among molecules of the same RNA component. Pairs of BMV RNA3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny RNA3 in a large number of lesions was analyzed. The majority of doubly infected lesions accumulated the RNA3 recombinants. The distribution of the recombinant types was relatively even, indicating that both RNA3 counterparts could serve as donor or as acceptor molecules. The frequency of crossovers between one pair of RNA3 variants, which possessed closely located markers, was similar to that of another pair of RNA3 variants with more distant markers, suggesting the existence of an internal recombination hot spot. The majority of crossovers were precise, but some recombinants had minor sequence modifications, possibly marking the sites of imprecise homologous crossovers. Our results suggest discontinuous RNA replication, with the replicase changing among the homologous RNA templates and generating RNA diversity. This approach can be easily extended to other RNA viruses for identification of homologous recombination hot spots.     

RNA determinants of junction site selection in RNA virus recombinants and defective interfering RNAs.

RNA recombination plays an important role in the diversification and evolution of RNA viruses. Most of these events are believed to be mediated by an actively copying viral replicase switching from a donor template to an acceptor template, where it resumes synthesis. In addition, intramolecular replicase-mediated events (i.e., rearrangements) can lead to the generation of replicable deleted forms of a viral genome, termed defective interfering (DI) RNAs. To gain further insight into the recombination process, the effect of various primary and secondary structures on recombination site selection in vivo was examined using plant RNA tombusviruses. The effect of sequence identity and complementarity on deletion events that generate DI RNAs was also investigated. Our results suggest that (1) 5' termini and strong hairpin structures in donor templates represent preferred sites for recombinations, (2) junction sites in acceptor templates do not occur in double-stranded regions, (3) nucleotide homology can shift donor and acceptor recombination sites closer to regions of identity and, (4) both sequence identity and complementarity can direct deletion sites in DI RNAs. These results further define RNA determinants of tombusvirus RNA recombination and rearrangement.     

RNA recombination in brome mosaic virus, a model plus strand RNA virus.

Studies on the molecular mechanism of genetic recombination in RNA viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. The system of brome mosaic virus (BMV) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of RNA-RNA recombination events. By using engineered BMV RNA components, the occurrence of both homologous and nonhomologous crosses were demonstrated among the segments of the BMV RNA genome. Studies show that the two types of crossovers require different RNA signal sequences and that both types depend upon the participation of BMV replicase proteins. Mutations in the two BMV-encoded replicase polypeptides (proteins 1a and 2a) reveal that their different regions participate in homologous and in nonhomologous crossovers. Based on all these data, it is most likely that homologous and nonhomologous recombinant crosses do occur via two different types of template switching events (copy-choice mechanism) where viral replicase complex changes RNA templates during viral RNA replication at distinct signal sequences. In this review we discuss various aspects of the mechanism of RNA recombination in BMV and we emphasize future projections of this research.     

Different mechanisms of homologous and nonhomologous recombination in brome mosaic virus: role of RNA sequences and replicase proteins

Brome mosaic bromovirus, a tripartite, positive-stranded RNA virus of plants, can generate both homologous and nonhomologous recombinantsin vivo. Recombination signals in the RNAs were different for these two recombination types. Nonhomologous recombination requires the formation of local double-stranded regions between the RNA templates. In contrast, homologous recombination is facilitated by AU-rich sequences and upstream GC-rich regions common in the recombining RNAs. Mutations within the replicase proteins affect homologous and nonhomologous recombination in different ways, confirming the involvement of BMV replicase proteins in both types of events as well as the differences in their pathways. Replicase-driven template-switching models are discussed in relation to supporting evidences.     

Emergence of distinct brome mosaic virus recombinants is determined by the polarity of the inoculum RNA

Despite overwhelming interest in the impact exerted by recombination during evolution of RNA viruses, the relative contribution of the polarity of inoculum templates remains poorly understood. Here, by agroinfiltrating Nicotiana benthamiana leaves, we show that brome mosaic virus (BMV) replicase is competent to initiate positive-strand [(+)-strand] synthesis on an ectopically expressed RNA3 negative strand [(-) strand] and faithfully complete the replication cycle. Consequently, we sought to examine the role of RNA polarity in BMV recombination by expressing a series of replication-defective mutants of BMV RNA3 in (+) or (-) polarity. Temporal analysis of progeny sequences revealed that the genetic makeup of the primary recombinant pool is determined by the polarity of the inoculum template. When the polarity of the inoculum template was (+), the recombinant pool that accumulated during early phases of replication was a mixture of nonhomologous recombinants. These are longer than the inoculum template length, and a nascent 3' untranslated region (UTR) of wild-type (WT) RNA1 or RNA2 was added to the input mutant RNA3 3' UTR due to end-to-end template switching by BMV replicase during (-)-strand synthesis. In contrast, when the polarity of the inoculum was (-), the progeny contained a pool of native-length homologous recombinants generated by template switching of BMV replicase with a nascent UTR from WT RNA1 or RNA2 during (+)-strand synthesis. Repair of a point mutation caused by polymerase error occurred only when the polarity of the inoculum template was (+). These results contribute to the explanation of the functional role of RNA polarity in recombination mediated by copy choice mechanisms.     

Silencing Homologous RNA Recombination Hot Spots with GC-Rich Sequences in Brome Mosaic Virus

It has been observed that AU-rich sequences form homologous recombination hot spots in brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants (P. D. Nagy and J. J. Bujarski, J. Virol. 71:3799–3810, 1997). To study the effect of GC-rich sequences on the recombination hot spots, we inserted 30-nucleotide-long GC-rich sequences downstream of AU-rich homologous recombination hot spot regions in parental BMV RNAs (RNA2 and RNA3). Although these insertions doubled the length of sequence identity in RNA2 and RNA3, the incidence of homologous RNA2 and RNA3 recombination was reduced markedly. Four different, both highly structured and nonstructured downstream GC-rich sequences had a similar “homologous recombination silencing” effect on the nearby hot spots. The GC-rich sequence-mediated recombination silencing mapped to RNA2, as it was observed when the GC-rich sequence was inserted at downstream locations in both RNA2 and RNA3 or only in the RNA2 component. On the contrary, when the downstream GC-rich sequence was present only in the RNA3 component, it increased the incidence of homologous recombination. In addition, upstream insertions of similar GC-rich sequences increased the incidence of homologous recombination within downstream hot spot regions. Overall, this study reveals the complex nature of homologous recombination in BMV, where sequences flanking the common hot spot regions affect recombination frequency. A replicase-driven template-switching model is presented to explain recombination silencing by GC-rich sequences.     

Role of RNA structure in non-homologous recombination between genomic molecules of brome mosaic virus

Brome mosaic virus (BMV) is a tripartite genome, positive-sense RNA virus of plants. Previously it was demonstrated that local hybridization between BMV RNAs (RNA–RNA heteroduplex formation) efficiently promotes non-homologous RNA recombination. In addition, studies on the role of the BMV polymerase in RNA recombination suggested that the location of non-homologous crossovers depends mostly on RNA structure. As a result, a detailed analysis of a large number of non-homologous recombinants generated in the BMV-based system was undertaken. Recombination hot-spots as well as putative elements in RNA structure enhancing non-homologous crossovers and targeting them in a site-specific manner were identified. To verify these observations the recombinationally active sequence in BMV RNA3 derivative was modified. The results obtained with new RNA3 mutants suggest that the primary and secondary structure of the sequences involved in a heteroduplex formation rather than the length of heteroduplex plays the most important role in the recombination process. The presented data indicate that the sequences proximal to the heteroduplex may also affect template switching by BMV replicase. Moreover, it was shown that both short homologous sequences and a hairpin structure have to accompany a double-stranded region to target non-homologous crossovers in a site-specific manner.     

Mutations in the helicase-like domain of protein 1a alter the sites of RNA-RNA recombination in brome mosaic virus.

A system that uses engineered heteroduplexes to efficiently direct in vivo crossovers between brome mosaic virus (BMV) RNA1 and RNA3 (P. Nagy and J. Bujarski, Proc. Natl. Acad. Sci. USA 90:6390-6394, 1993) has been used to explore the possible involvement of BMV 1a protein, an essential RNA replication factor, in RNA recombination. Relative to wild-type 1a, several viable amino acid insertion mutations in the helicase-like domain of BMV 1a protein affected the nature and distribution of crossover sites in RNA3-RNA1 recombinants. At 24 degrees C, mutants PK19 and PK21 each increased the percentage of asymmetric crossovers, in which the RNA1 and RNA3 sites joined by recombination were not directly opposite each other on the engineered RNA3-RNA1 heteroduplex used to target recombination but rather were separated by 4 to 85 nucleotides. PK21 and another 1a mutant, PK14, also showed increases in the fraction of recombinants containing nontemplated U residues at the recombination junction. At 33 degrees C, the highest temperature that permitted infections with PK19, which is temperature sensitive for RNA replication, the mean location of RNA1-RNA3 crossovers in recombinants recovered from PK19 infections was shifted by nearly 25 bp into the energetically less stable side of the RNA1-RNA3 heteroduplex. Thus, mutations in the putative helicase domain of the 1a protein can influence BMV RNA recombination. The results are discussed in relation to models for recombination by template switching during pausing of RNA replication at a heteroduplexed region in the template.     

Nonhomologous RNA recombination during negative-strand synthesis of flock house virus RNA.

During sequential replicative passages of viral RNA from the nodavirus flock house virus, spontaneous deletion of RNA sequences occurred frequently. Families of deleted RNA molecules were derived from both segments of the bipartite viral genome and found to contain single, double, or triple deletions. These deletions were attributed to template switching by the flock house virus RNA replicase, resulting in recombination between distant sequences and excision of the intervening nucleotides. From sequence analysis of the recombination junctions, we concluded that the process of template switching was influenced by both the primary sequence and the secondary structure of the RNA and that it occurred predominantly during synthesis of RNA negative strands.     

Viral RNA-directed RNA polymerases use diverse mechanisms to promote recombination between RNA molecules

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Qbeta phage-derived RNA, such that if aligned at complementary 3'-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Qbeta replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Qbeta replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Qbeta RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qbeta replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qbeta phage.     

Efficient system of homologous RNA recombination in brome mosaic virus: sequence and structure requirements and accuracy of crossovers.

Brome mosaic virus (BMV), a tripartite positive-stranded RNA virus of plants engineered to support intersegment RNA recombination, was used for the determination of sequence and structural requirements of homologous crossovers. A 60-nucleotide (nt) sequence, common between wild-type RNA2 and mutant RNA3, supported efficient repair (90%) of a modified 3' noncoding region in the RNA3 segment by homologous recombination with wild-type RNA2 3' noncoding sequences. Deletions within this sequence in RNA3 demonstrated that a nucleotide identity as short as 15 nt can support efficient homologous recombination events, while shorter (5-nt) sequence identity resulted in reduced recombination frequency (5%) within this region. Three or more mismatches within a downstream portion of the common 60-nt RNA3 sequence affected both the incidence of recombination and the distribution of crossover sites, suggesting that besides the length, the extent of sequence identity between two recombining BMV RNAs is an important factor in homologous recombination. Site-directed mutagenesis of the common sequence in RNA3 did not reveal a clear correlation between the stability of predicted secondary structures and recombination activity. This indicates that homologous recombination does not require similar secondary structures between two recombining RNAs at the sites of crossovers. Nearly 20% of homologous recombinants were imprecise (aberrant), containing either nucleotide mismatches, small deletions, or small insertions within the region of crossovers. This implies that homologous RNA recombination is not as accurate as proposed previously. Our results provide experimental evidence that the requirements and thus the mechanism of homologous recombination in BMV differ from those of previously described heteroduplex-mediated nonhomologous recombination (P. D. Nagy and J. J. Bujarski, Proc. Natl. Acad. Sci. USA 90:6390-6394, 1993).     

Nonhomologous RNA-RNA recombination events at the 3' nontranslated region of the Sindbis virus genome: hot spots and utilization of nonviral sequences.

The mechanism of RNA-RNA recombination at the 3' nontranslated region (3'NTR) of the Sindbis virus (SIN) genome was studied by using nonreplicative RNA precursors. The 11.7-kb SIN genome was transcribed in vitro as two nonoverlapping RNA fragments. RNA-1 contained the entire 11.4-kb protein coding sequence of SIN and also carried an additional 1.8-kb nonviral sequence at its 3' end. RNA-2 carried the remaining 0.26 or 0.3 kb of the SIN genome containing the 3'NTR. Transfection of these two fragments into BHK cells resulted in vivo RNA-RNA recombination and release of infectious SIN recombinants. Eighteen plaque-purified recombinant viruses were sequenced to precisely map the RNA-RNA crossover sites at the 3'NTR. Sixteen of the 18 recombinants were found to be genetically heterogeneous at the 3'NTR. Two major clustered sites within the 3'NTR of RNA-2 were found to be fused to multiple locations on the nonviral sequence of RNA-1, resulting in insertions of 10 to 1,085 nucleotides at the 3'NTR. Sequence analysis of crossover sites suggested only limited homology and heteroduplex-forming capability between substrate RNAs. Analysis of additional 23 recombinant viruses generated by mutagenized donor and acceptor templates supports the occurrence of recombination hot spots on donor templates. Introduction of a 17-nucleotide rudimentary replicase recognition signal in the acceptor template alone did not induce the polymerase to reinitiate at the 17-nucleotide signal. Interestingly, deletion of a 24-nucleotide hot spot locus on the donor template abolished crossover events at one of the two sites and allowed the polymerase to reinitiate at the 17-nucleotide replicase recognition signal inserted at the acceptor template. The possible roles of RNA-protein and RNA-RNA interactions in the differential regulation of apparent pausing, template selection, and reinitiation are discussed.     

Inhibition of RNA Recruitment and Replication of an RNA Virus by Acridine Derivatives with Known Anti-Prion Activities

Background

Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV), a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ) and quinacrine (QC), which are active against prion-based diseases.

Methodology/Principal Findings

Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i) inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii) reduction of minus-strand synthesis by the tombusvirus replicase; and (iii) inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication.

Conclusion/Significance

Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses.     

Homologous RNA recombination in brome mosaic virus: AU-rich sequences decrease the accuracy of crossovers.

Brome mosaic virus, a tripartite positive-stranded RNA virus of plants, was used for the determination of sequence requirements of imprecise (aberrant) homologous recombination. A 23-nucleotide (nt) region that included a 6-nt UUAAAA sequence (designated the AU sequence) common between wild-type RNA2 and mutant RNA3 supported both precise and imprecise homologous recombination, though the latter occurred with lower frequency. Doubling the length of the 6-nt AU sequence in RNA3 increased the incidence of imprecise crossovers by nearly threefold. Duplication or triplication of the length of the AU sequence in both RNA2 and RNA3 further raised the frequency of imprecise crossovers. The majority of imprecise crosses were located within or close to the extended AU sequence. Imprecise recombinants contained either nucleotide substitutions, nontemplated nucleotides, small deletions, or small sequence duplications within the region of crossovers. Deletion of the AU sequence from the homologous region in RNA3 resulted in the accumulation of only precise homologous recombinants. Our results provide experimental evidence that AU sequences can facilitate the formation of imprecise homologous recombinants. The generation of small additions or deletions can be explained by a misannealing mechanism within the AU sequences, while replicase errors during RNA copying might explain the occurrence of nucleotide substitutions or nontemplated nucleotides.     

Mutations in the antiviral RNAi defense pathway modify Brome mosaic virus RNA recombinant profiles

RNA interference (RNAi) mechanism targets viral RNA for degradation. To test whether RNAi gene products contributed to viral RNA recombination, a series of Arabidopsis thaliana RNAi-defective mutants were infected with Brome mosaic virus (BMV) RNAs that have been engineered to support crossovers within the RNA3 segment. Single-cross RNA3-RNA1, RNA3-RNA2, and RNA3-RNA3 recombinants accumulated in both the wild-type (wt) and all knock-out lines at comparable frequencies. However, a reduced accumulation of novel 3' mosaic RNA3 recombinants was observed in ago1, dcl2, dcl4, and rdr6 lines but not in wt Col-0 or the dcl3 line. A BMV replicase mutant accumulated a low level of RNA3-RNA1 single-cross recombinants in Col-0 plants while, in a dcl2 dcl4 double mutant, the formation of both RNA3-RNA1 and mosaic recombinants was at a low level. A control infection in the cpr5-2 mutant, a more susceptible BMV Arabidopsis host, generated similar-to-Col-0 profiles of both single-cross and mosaic recombinants, indicating that recombinant profiles were, to some extent, independent of a viral replication rate. Also, the relative growth experiments revealed similar selection pressure for recombinants among the host lines. Thus, the altered recombinant RNA profiles have originated at the level of recombinant formation rather than because of altered selection. In conclusion, the viral replicase and the host RNAi gene products contribute in distinct ways to BMV RNA recombination. Our studies reveal that the antiviral RNAi mechanisms are utilized by plant RNA viruses to increase their variability, reminiscent of phenomena previously demonstrated in fungi.