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ABSTRACTA 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.

ABSTRACTBrome 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.

We describe spontaneous second-site reversions within the coat protein open reading frame that rescue the systemic-spread phenotype and increase virion stability of a mutant of brome mosaic virus. Based on the crystal structure of the related cowpea chlorotic mottle virus, we show that the modified residues are spatially clustered to affect the formation of hexamers and pentamers and therefore virion stability.

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.

An in vitro transcription system has been developed that utilizes self-processing to adjust 3' termini in transcribed viral RNAs. Transcription vectors were constructed by ligating a cDNA "cassette" containing self-cleavage sequences from the satellite RNA of tobacco ringspot virus (STobRV RNA) with the 3' end of brome mosaic virus (BMV) cDNA clones. Transcripts were obtained either from templates linearized at positions located downstream from the cassette or from nonlinearized plasmids. In both cases, a spontaneous self-cleavage reaction produced BMV RNAs that contained 19 heterologous nucleotides at their 3' ends. These RNAs were biologically active, indicating that 19 extra nucleotides did not remove the infectivity. The progeny BMV RNA isolated from infected plants did not retain the 19-base heterologous sequence. BMV RNAs containing longer 3' heterologous sequences were not infectious. The STobRV self-cleavage cassette is potentially applicable to a variety of RNA systems.

Deletions in cDNA clones covering the 3' 201 nucleotides of brome mosaic virus RNA 3 were produced by S1 nuclease treatment of cloned DNA linearized at several different restriction sites. Transcription of these clones yielded RNAs containing structural alterations in the 3'-terminal tRNA-like structure that is involved in aminoacylation and replication. Replicase template activity, but not aminoacylation activity, was especially sensitive to deletions in arm C, which contains a tyrosyl anticodon. Deletions in arm B were detrimental to aminoacylation, but the proportion of replicase template activity lost depended on the site of the deletion. Removal of arm D had little effect on aminoacylation and, in some instances, resulted in a 2-fold stimulation of replicase template activity.

ABSTRACTPreviously, we have observed that mutations in proteins 1a and 2a, the two virally encoded components of the brome mosaic virus (BMV) replicase, can affect the frequency of recombination and the locations of RNA recombination sites (P. D. Nagy, A. Dzianott, P. Ahlquist, and J. J. Bujarski, J. Virol. 69:2547–2556, 1995; M. Figlerowicz, P. D. Nagy, and J. J. Bujarski, Proc. Natl. Acad. Sci. USA 94:2073–2078, 1997). Also, it was found before that the N-terminal domain of 2a, the putative RNA polymerase protein, participates in the interactions between 1a and 2a (C. C. Kao, R. Quadt, R. P. Hershberger, and P. Ahlquist, J. Virol. 66:6322–6329, 1992; E. O’Reilly, J. Paul, and C. C. Kao, J. Virol. 71:7526–7532, 1997). In this work, we examine how mutations within the N terminus of 2a influence RNA recombination in BMV. Because of the likely electrostatic character of 1a-2a interactions, five 2a mutants, MF1 to MF5, were generated by replacing clusters of acidic amino acids with their neutral counterparts. MF2 and MF5 retained nearly wild-type levels of 1a-2a interaction and were infectious inChenopodium quinoa. However, compared to that in wild-type virus, the frequency of nonhomologous recombination in both MF2 and MF5 was markedly decreased. Only in MF2 was the frequency of homologous recombination reduced and the occurrence of imprecise homologous recombination increased. In MF5 there was also a 3' shift in the positions of homologous crossovers. The observed effects of MF2 and MF5 reveal that the 2a N-terminal domain participates in different ways in homologous and in nonhomologous BMV RNA recombination. This work maps specific locations within the N terminus involved in 1a-2a interaction and in recombination and further suggests that the mechanisms of the two types of crossovers in BMV are different.

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.

A remarkable feature of the genomic RNAs of several plant viruses is the presence at the 3′ end of a region that exhibits tRNA-like functions, including aminoacylation1–3. The three genomic and single subgenomic RNAs of brome mosaic virus (BMV) accept tyrosine in vitro4and in vivo5, the smallest 3′ fragment that can be aminoacylated being about 135 nucleotides long6. The roles of the tRNA-like properties are incompletely understood, but an involvement in replication rather than translational functions is likely3,7,8. We have recently shown (J.J.B. et al., in preparation) that the features recognized by the BMV RNA-specific RNA-dependent RNA polymerase (replicase)9,10for the use of BMV RNA for complementary strand synthesis also lie within the tRNA-like structure. To distinguish between the roles of BMV RNA as a substrate for tyrosyl-tRNA synthetase and BMV replicase, we have now produced BMV RNAs with mutations at two separate loci within the tRNA-like structure. This has been achieved by transcription in vitro from specifically mutagenized cDNA, an approach permitting the generation of targeted mutants without regard to their viability in vivo.

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).

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.

All three single-stranded RNAs of the brome mosaic virus (BMV) genome contain a highly conserved, 193-base 3' noncoding region. To study the recombination between individual BMV RNA components, barley plants were infected with a mixture of in vitro-transcribed wild-type BMV RNAs 1 and 2 and an RNA3 mutant that carried a deletion near the 3' end. This generated a population of both homologous and nonhomologous 3' recombinant BMV RNA3 variants. Sequencing revealed that these recombinants were derived by either single or double crossovers with BMV RNA1 or RNA2. The primary sequences at recombinant junctions did not show any similarity. However, they could be aligned to form double-stranded heteroduplexes. This suggested that local hybridizations among BMV RNAs may support intermolecular exchanges.

It has been postulated that local hybridizations between viral RNAs can mediate recombination in brome mosaic virus (BMV) and in poliovirus. To test this model, a 3' fragment of BMV RNA1 was inserted into the 3' noncoding sequence of BMV RNA3 in an antisense orientation. This resulted in high-frequency nonhomologous crossovers at or near the hybridized region. Insertion of the same RNA1 fragment in a positive-sense orientation did not promote recombination. Modification of the antisense insert by deletion of 3' portions did not affect the sites of crossover. However, modification of the 5' portion shifted the crossovers toward the central part of the heteroduplex region. Our results provide experimental evidence that recombinant crosses can be primed by hybridization between viral RNA molecules.

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.