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.     

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).     

Homologous crossovers among molecules of brome mosaic bromovirus RNA1 or RNA2 segments in vivo

Previously we demonstrated frequent homologous crossovers among molecules of the RNA3 segment in the tripartite brome mosaic bromovirus (BMV) RNA genome (A. Bruyere, M. Wantroba, S. Flasinski, A. Dzianott, and J. J. Bujarski, J. Virol. 74:4214-4219, 2000). To further our knowledge about mechanisms of viral RNA genome variability, in this paper we have studied homologous recombination in BMV RNA1 and RNA2 components during infection. We have found that basal RNA-RNA crossovers could occur within coding regions of both RNAs, although recombination frequencies slightly varied at different RNA sections. In all cases, the frequencies were much lower than the rate observed for the intercistronic recombination hot spot in BMV RNA3. Probability calculations accounted for at least one homologous crossover per RNA molecule per replication cycle. In addition, we have demonstrated an efficient repair of mutations within the conserved 3' and 5' noncoding regions, most likely due to error-prone BMV RNA replication. Overall, our data verify that homologous crossovers are common events a during virus life cycle, and we discuss their importance for viral RNA genetics.     

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.     

Two types of non-homologous RNA recombination in brome mosaic virus

Non-homologous RNA recombination is a process enabling the exchange of genetic material between various (related or unrelated) RNA-based viruses. Despite extensive investigations its molecular mechanism remains unclear. Studies on genetic recombination in brome mosaic virus (BMV) have shown that local hybridization between genomic RNAs induces frequent non-homologous crossovers. A detailed analysis of recombinant structures suggested that local complementary regions might be involved in two types of non-homologous recombination in BMV: site-specific and heteroduplex-mediated. To verify the above hypothesis and better recognize the mechanism of the phenomenon studied we have tested how the putative types of recombination are affected by a specific mutation in the BMV polymerase gene or by changes in RNA structure. The experiments undertaken revealed substantial differences between site-specific and heteroduplex-mediated recombination, indicating that they occur according to different mechanisms. The former can be classified as homology-assisted, and the latter as homology-independent. In addition to local RNA/RNA hybridization, short regions of homology are required for site-specific crossovers to occur. They are most efficiently mediated if one homologous sequence is located at the beginning of and the second just before a double-stranded region. At present it is difficult to state what is the mechanism of heteroduplex-mediated recombination. Earlier it was postulated that strong RNA/RNA interaction enforces template switching by the viral replicase. There are, however, several observations questioning this model and indicating that some other factors, which are still unknown, may influence heteroduplex-mediated crossovers.     

RNA recombination in brome mosaic virus: effects of strand-specific stem-loop inserts

A model system of a single-stranded trisegment Brome mosaic bromovirus (BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3' noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive- rather than negative-strand synthesis.     

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.     

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.     

Comparison of the nucleotide sequences of cucumber mosaic virus and brome mosaic virus

Cucumber mosaic virus (CMV) and brome mosaic virus (BMV) are isometric plant viruses. Although biologically distinct, they share many common chemical properties. An analysis of the partial genomic RNA sequence available for these two viruses reveals that they are evolutionarily related. Different segments of the genome exhibit different evolutionary rates. The coat proteins, which serve as carriers of genetic material, possess little or no homology. In contrast, the 3a proteins show over 35% homology. The non-coding regions of the genome also exhibit extensive but variable homology suggesting the functional importance of the nucleic acid.     

Characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus subgenomic RNA.

Expression of brome mosaic virus (BMV) coat protein and internal genes of many other positive-strand RNA viruses requires initiation of subgenomic mRNA synthesis from specific internal sites on minus-strand genomic RNA templates. Biologically active viral cDNA clones were used to investigate the sequences controlling production of BMV subgenomic RNA in vivo. Suitable duplications directed production of specifically initiated, capped subgenomic RNAs from new sites in the BMV genome. Previously implicated promoter sequences extending 20 bases upstream (-20) and 16 bases downstream (+16) of the subgenomic RNA initiation site directed only low-level synthesis. Subgenomic RNA production at normal levels required sequences extending to at least -74 but not beyond -95. Loss of an (rA)18 tract immediately upstream of the -20 to +16 "core promoter" particularly inhibited subgenomic RNA synthesis. The -38 to -95 region required for normal initiation levels contains repeats of sequence elements in the core promoter, and duplications creating additional upstream copies of these repeats stimulated subgenomic RNA synthesis above wild-type levels. At least four different subgenomic RNAs can be produced from a single BMV RNA3 derivative. For all derivatives producing more than one subgenomic RNA, a gradient of accumulation progressively favoring smaller subgenomic RNAs was seen.     

The AU-rich RNA recombination hot spot sequence of Brome mosaic virus is functional in tombusviruses: implications for the mechanism of RNA recombination

RNA recombination can be facilitated by recombination signals present in viral RNAs. Among such signals are short sequences with high AU contents that constitute recombination hot spots in Brome mosaic virus (BMV) and retroviruses. In this paper, we demonstrate that a defective interfering (DI) RNA, a model template associated with Tomato bushy stunt virus (TBSV), a tombusvirus, undergoes frequent recombination in plants and protoplast cells when it carries the AU-rich hot spot sequence from BMV. Similar to the situation with BMV, most of the recombination junction sites in the DI RNA recombinants were found within the AU-rich region. However, unlike BMV or retroviruses, where recombination usually occurred with precision between duplicated AU-rich sequences, the majority of TBSV DI RNA recombinants were imprecise. In addition, only one copy of the AU-rich sequence was essential to promote recombination in the DI RNA. The selection of junction sites was also influenced by a putative cis-acting element present in the DI RNA. We found that this RNA sequence bound to the TBSV replicase proteins more efficiently than did control nonviral sequences, suggesting that it might be involved in replicase "landing" during the template switching events. In summary, evidence is presented that a tombusvirus can use the recombination signal of BMV. This supports the idea that common AU-rich recombination signals might promote interviral recombination between unrelated viruses.     

Genetic recombination in brome mosaic virus: effect of sequence and replication of RNA on accumulation of recombinants.

In order to facilitate the isolation of recombinants in brome mosaic virus, a series of duplication mutants with alterations in the RNA3 3' noncoding region has been engineered. The distribution of crossovers, which was observed to be dependent on the parental RNA3 sequence, supported the role of RNA structure in recombination. However, a negative correlation between replication of the parental RNA3 constructs and the accumulation of recombinant progeny confirmed the role of selection.     

Intercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA3.

The genome of brome mosaic virus (BMV) is divided among messenger polarity RNA1, RNA2, and RNA3 (3.2, 2.9, and 2.1 kilobases, respectively). cis-Acting sequences required for BMV RNA amplification were investigated with RNA3. By using expressible cDNA clones, deletions were constructed throughout RNA3 and tested in barley protoplasts coinoculated with RNA1 and RNA2. In contrast to requirements for 5'- and 3'-terminal noncoding sequences, either of the two RNA3 coding regions can be deleted individually and both can be simultaneously inactivated by N-terminal frameshift mutations without significantly interfering with amplification of RNA3 or production of its subgenomic mRNA. However, simultaneous major deletions in both coding regions greatly attenuate RNA3 accumulation. RNA3 levels can be largely restored by insertion of a heterologous, nonviral sequence in such mutants, suggesting that RNA3 requires physical separation of its terminal domains or a minimum overall size for normal replication or stability. Unexpectedly, deletions in a 150-base segment of the intercistronic noncoding region drastically reduce RNA3 accumulation. This segment contains a sequence element homologous to sequences found near the 5' ends of BMV RNA1 and RNA2 and in analogous positions in the three genomic RNAs of the related cucumber mosaic virus, suggesting a possible role in plus-strand synthesis.     

Stable RNA structures can repress RNA synthesis in vitro by the brome mosaic virus replicase

A 15-nucleotide (nt) unstructured RNA with an initiation site but lacking a promoter could direct the initiation of RNA synthesis by the brome mosaic virus (BMV) replicase in vitro. However, BMV RNA with a functional initiation site but a mutated promoter could not initiate RNA synthesis either in vitro or in vivo. To explain these two observations, we hypothesize that RNA structures that cannot function as promoters could prevent RNA synthesis by the BMV RNA replicase. We documented that four different nonpromoter stem-loops can inhibit RNA synthesis from an initiation-competent RNA sequence in vitro. Destabilizing these structures increased RNA synthesis. However, RNA synthesis was restored in full only when a BMV RNA promoter element was added in cis. Competition assays to examine replicase-RNA interactions showed that the structured RNAs have a lower affinity for the replicase than do RNAs lacking stable structures or containing a promoter element. The results characterize another potential mechanism whereby the BMV replicase can specifically recognize BMV RNAs.     

Localization of the replicase recognition site within brome mosaic virus RNA by hybrid-arrested RNA synthesis

Summary 3 terminal fragments of BMV RNA as short as 153 bases in length serve as efficient templates in vitro for BMV-specific RNA polymerase. Template activity of such fragments or of native BMV RNA is abolished when cDNA fragments as short as 39 bases are hybridized to their 3 termini. Hybridization of cDNa fragments to regions of BMV RNA 200 or more bases distal to the 3 end has no discernible effect on initiation and little effect on elongation. We conclude that BMV RNA polymerase initiates binding with an RNA template through a mechanism mediated by the tRNA-like 3 end of BMV RNA, requiring at least some of the last 39, but no more than the last 153 bases.