Genetic RNA recombination plays an important role in viral evolution, but its molecular mechanism is not well understood. In this work we describe homologous RNA recombination activity that is supported by a subgenomic promoter (sgp) region in the RNA3 segment of brome mosaic bromovirus (BMV), a tripartite plusstrand RNA virus. The crossover frequencies were determined by coinoculations with pairs of BMV RNA3 variants that carried a duplicated sgp region flanked by marker restriction sites. A region composed of the sgp core, a poly(A) tract, and an upstream enhancer supported homologous exchanges in 25% of the analyzed RNA3 progeny. However, mutations in the sgp core stopped both the transcription of the sgp RNA and homologous recombination. These data provide evidence for an association of RNA recombination with transcription.Genetic recombination is an important process leading to diversity among living organisms. The mechanisms of crossing over (general homologous recombination) and other routes of genetic exchange are relatively well studied in DNA-based organisms. In contrast, although RNA recombination has been studied extensively for several RNA virus groups, including bromoviruses, picornaviruses, coronaviruses, tombusviruses, and bacteriophages (8), its mechanism is not well understood (8, 13). Brome mosaic bromovirus (BMV) is a tripartite RNA virus where RNA components 1 and 2 encode, respectively, the replicase proteins 1a and 2a, while RNA3 encodes the movement (3a) and the coat proteins (CPs) (3). CP is expressed from a subgenomic (sg) RNA4.The subgenomic promoter (sgp) for RNA4 is located internally on the minus strand of BMV RNA3. It includes a core region that is responsible for binding of the viral RNA polymerase (RdRp) and the initiation of transcription (30), an "enhancing" region, a poly(A) stretch, and a downstream portion (21, 34). Besides BMV, the multielement nature of sgp's has been demonstrated in other RNA viruses (24).There is only limited information about recombination events in natural populations of viral RNA molecules. Recombination hot spots were observed during the course of infection in poliovirus (16) and in human immunodeficiency virus type 1 (36). It has been proposed that recombination within the sg RNA start sites has led to the formation of genera of luteoviruses (23) or has served as a factor for the modular exchange and rearrangements of the genomes of closteroviruses (5). Also, the recombination hot spots are thought to be associated with RNA replication enhancers, such as those found in turnip crinkle carmovirus (9, 30). RNA structure has been reported to play a role in promoting RNA crossovers in retroviruses (4).In BMV, the frequency of homologous intersegmental crossovers is approximately 10 times higher than that of nonhomologous crossovers (27). A model of the replicase template switching has been proposed to explain the observed homologous crossovers between BMV RNAs (28, 29). An increased recombination activity has been demonstrated within the intercistronic r...

. In order to correlate sgp-mediated recombination and transcription, in the present work we used BMV RNA3 constructs that carried altered sgp repeats. We observed that the removal or extension of the poly(U) tract reduced or increased recombination, respectively. Deletion of the sgp core hairpin or its replacement by a different stem-loop structure inhibited recombination activity. Nucleotide substitutions at the ؉1 or ؉2 transcription initiation position reduced recombination. The sgp core alone supported only basal recombination activity. The sites of crossovers mapped to the poly(U) region and to the core hairpin. The observed effects on recombination did not parallel those observed for transcription. To explain how both activities operate within the sgp sequence, we propose a dual mechanism whereby recombination is primed at the poly(U) tract by the predetached nascent plus strand, whereas transcription is initiated de novo at the sgp core.Viral RNA recombination plays an important role during rearrangements of viral RNAs and provides an efficient tool for the repair of their genomic sequences (12, 12A, 36, 40). In a variety of RNA viruses, including the bromovirus brome mosaic virus (BMV), coronaviruses, poliovirus, carmoviruses, tombusviruses, and flaviviruses (7, 8), RNA recombination events seem to occur via a copy choice mechanism, where the replicase enzyme changes templates during RNA synthesis. The evidence is based on the effects of RNA sequence modifications and the participation of replicase proteins (49) in recombination, both in vivo and in vitro (14,16,18,53). In retroviruses, there seem to be three, non-mutually exclusive copy choice mechanisms: forced (strong-stop) strand transfer, pause-driven strand transfer, and pause-independent (RNA structure-driven) strand transfer (26). Other proposed mechanisms are cleavage-religation (20, 37, 40) and transesterification (12, 12A, 20).BMV is a tripartite RNA virus, where RNA components 1 and 2 (RNA1 and RNA2) encode, respectively, replicase proteins 1a and 2a, while RNA3 encodes a movement protein (3a) and a coat protein (CP) (4). The CP is expressed from subgenomic (sg) RNA4.In BMV, the frequency of homologous intersegmental recombination within the 3Ј noncoding region is approximately 10 times higher than that of nonhomologous crossovers (17,38). Most of the 3Ј crossovers are precise (38, 39) and are concentrated within GC-rich sequences followed by downstream AU-rich regions (38-40). The imprecise crossovers pinpoint the actual crossover sites. A proposed model suggests that the BMV RNA-dependent RNA polymerase (RdRp) pauses (stalls) at AU-rich sequences and then switches onto the acceptor template, with the upstream GC-rich domain facilitating the rehybridization of the detached nascent strand (39,40).Besides recombination among different RNA segments, homologous recombination activity between RNA3 molecules has been mapped to the intercistronic region (11). This consists of the subgenomic promoter (sgp) on the minus strand of RNA3 (32) and the 1...

The synthesis of 3 subgenomic RNA4 (sgRNA4) by initiation from an internal sg promoter in the RNA3 segment was first described for Brome mosaic bromovirus (BMV), a model tripartite positive-sense RNA virus (W. A. Miller, T. W. Dreher, and T. C. Hall, Nature 313: [68][69][70] 1985). In this work, we describe a novel 5 sgRNA of BMV (sgRNA3a) that we propose arises by premature internal termination and that encapsidates in BMV virions. Cloning and sequencing revealed that, unlike any other BMV RNA segment, sgRNA3a carries a 3 oligo(A) tail, in which respect it resembles cellular mRNAs. Indeed, both the accumulation of sgRNA3a in polysomes and the synthesis of movement protein 3a in in vitro systems suggest active functions of sgRNA3a during protein synthesis. Moreover, when copied in the BMV replicase in vitro reaction, the minus-strand RNA3 template generated the sgRNA3a product, likely by premature termination at the minus-strand oligo(U) tract. Deletion of the oligo(A) tract in BMV RNA3 inhibited synthesis of sgRNA3a during infection. We propose a model in which the synthesis of RNA3 is terminated prematurely near the sg promoter. The discovery of 5 sgRNA3a sheds new light on strategies viruses can use to separate replication from the translation functions of their genomic RNAs.Single-stranded positive-sense RNA viruses utilize various strategies for expression of their RNA genomes (36), most notably via subgenomic RNAs (sgRNAs). The Coronaviridae and Arteriviridae families of the order Nidovirales express 6 to 7 proteins via sgRNAs, while the Closteroviridae generate between 6 and 11 sgRNAs. In some viruses, e.g., in Brome mosaic bromovirus (BMV), sgRNAs arise via internal initiation by the viral RNA polymerase (RdRp) on genomic minus-sense RNAs (20,30,33,38). It is also likely that other viruses, e.g., red clover necrotic mosaic virus (54), tomato bushy stunt virus (10), and flock house virus (17), copy their sgRNAs from prematurely terminated minus strands (62). In Nidovirales (46, 61, 72) the noncontiguous RNA leaders are joined to variously located sequences during minus-strand synthesis, followed by sgRNA transcription, whereas toroviruses combine discontinuous and nondiscontinuous processes to produce their sgRNAs (63). The formation of sgRNAs can also result from premature termination of positive-strand synthesis. In closteroviruses, a highly structured sequence region produces 5Ј-terminal sgRNAs by pretermination (20) and additionally serves as a promoter for synthesis of another downstream sgRNA, with possible overlapping of termination and initiation signals (20).The previously described 3Ј sgRNA4 of BMV is transcribed from an intergenic 100-nucleotide (nt) promoter (sgp) that consists of the core domain, a transcription enhancer, and the poly(A) tract (18,38,57). The more-upstream 150-nt sequence functions in cis as an internal replication enhancer (IRE) for positive-strand RNA3 amplification and carries a conserved B-box motif (18). In addition, we recently reported that the poly(A) tract of the sgp...

The sequences required for integration of retroviral DNA have been analyzed in vitro. However, the in vitro experiments do not agree on which sequences are required for integration: for example, whether or not the conserved CA dinucleotide in the 3 end of the viral DNA is required for normal integration. At least a portion of the problem is due to differences in the experimental conditions used in the in vitro assays. To avoid the issue of what experimental conditions to use, we took an in vivo approach. We made mutations in the 5 end of the U3 sequence of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z. We present evidence that, in RSV, the CA dinucleotide in the 5 end of U3 is not essential for appropriate integration. This result differs from the results seen with mutations in the U5 end, where the CA appears to be essential for proper integration in vivo. In addition, based on the structure of circular viral DNAs smaller than the full-length viral genome, our results suggest that there is little, if any, integrase-mediated autointegration of RSV linear DNA in vivo.The retroviral life cycle is characterized by the conversion of the single-stranded RNA genome found in virions into a double-stranded linear DNA that is subsequently integrated into the host cell genome. Viral DNA synthesis takes place in the cytoplasm of the infected cell. The RNA genome of the virus is copied into DNA by a virally encoded enzyme, reverse transcriptase (RT). RT contains two enzymatic activities, a DNA polymerase that can copy either an RNA or a DNA template, and an RNase H that cleaves RNA only if the RNA is part of an RNA/DNA hybrid. Like many other DNA polymerases, RT requires both a template and a primer. First (minus)-strand DNA synthesis is initiated from a cellular tRNA primer that is base paired at the primer binding site (PBS), which is near the 5Ј end of the viral genomic RNA. During the reverse transcription process, this tRNA primer is removed from the end of the minus-strand DNA by RNase H; this defines the right (U5) end of the linear viral DNA. Second (plus)-strand DNA synthesis is initiated from a polypurine tract (PPT) primer adjacent to U3. The RNase H cleavages that generate and remove PPT primer define the left (

Recent in vivo studies have revealed that the subgenomic promoter (sgp) in brome mosaic bromovirus (BMV) RNA3 supports frequent homologous recombination events (R. Wierzchoslawski, A. Dzianott, and J. Bujarski, J. Virol. 78: [8552][8553][8554][8555][8556][8557][8558][8559][8560][8561][8562][8563][8564] 2004). In this paper, we describe an sgp-driven in vitro system that supports efficient RNA3 crossovers. A 1:1 mixture of two (؊)-sense RNA3 templates was copied with either a BMV replicase (RdRp) preparation or recombinant BMV protein 2a. The BMV replicase enzyme supported a lower recombination frequency than 2a, demonstrating a role of other viral and/or host factors. The described in vitro system will allow us to study the mechanism of homologous RNA recombination.RNA recombination is one of the fundamental aspects of the life cycle and evolution of RNA viruses and contributes significantly to a high level of variation in viral RNAs, as demonstrated by sequencing of virus populations (6,7,32,34,45,50,58) and with experimental in vivo systems for animal viruses (19, 31, 44, 51, 54), plant viruses (1, 2, 12, 15, 25, 35, 41, 53, 55, 59), bacteriophages (38), and retroviruses (40). In vitro recombination assays (reviewed in reference 21) revealed either a nonreplicative (breakage-religation) (22) or replicative (template switch) (3, 28, 30, 36) mechanism of crossover. During the template switch, a premature dissociation of the replicating enzyme from the donor RNA molecule is evidently facilitated by double-stranded structures (9, 10, 30, 43, 51), homopolymeric tracts (11), AU-rich motifs (29), or subgenomic promoters (23). The detached replicase then reinitiates synthesis on the acceptor RNA, either de novo, usually in the vicinity of promoters or enhancers (13,14,49,51), or via priming by the nascent 3Ј end of the newly synthesized RNA (43).Brome mosaic bromovirus (BMV) is a tripartite RNA virus with frequent homologous crossovers within the subgenomic promoter (sgp) region of the RNA3 segment (5,20,31,47). The junctions cluster within the sgp poly(A) tract and the core, plausibly due to replicase detachment during copying of (Ϫ) strands (56,57). Mutagenesis of the sgp demonstrated a partial overlap of recombination and transcription activities in vivo (56, 57), and end-to-end template switching events have been described for BMV RNA-dependent RNA synthesis in vitro (28). In this paper, we present the first sgp-mediated RNA virus homologous recombination system, where BMV RNA3 crossovers occur by copying with either BMV replicase isolated from virus-infected plants or Escherichia coli-expressed protein 2a, which is a catalytic subunit of the BMV replicase. The recombination frequencies and junction sites varied between the two enzymes, leading to assumptions about the mechanism of crossover.Plasmid pB3TP7 (27) was used to prepare two similar RW (Ϫ) RNA3 constructs (Fig.