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.It is generally accepted that RNA recombination contributes significantly to the diversity of viruses with RNA genomes (37). However, little experimental evidence supports the occurrence of high-frequency recombination in the virus life cycle. The processes of RNA replication and RNA recombination have been studied extensively in brome mosaic bromovirus (BMV), a tripartite positive-strand RNA virus (12). BMV RNA1 and RNA2 code, respectively, for the 1a and 2a proteins (the viral components of the replicase complex), while RNA3 encodes the 3a (movement) and coat proteins (2). Both homologous and nonhomologous recombination events have been observed among different BMV RNAs (24). Homology-supported crossovers can occur between two nearly identical RNAs (or within nearly identical regions), while nonhomologous crosses can occur between nonrelated RNAs or dissimilar regions (8,16,29). The frequency of homologous intersegmental crosses in BMV is approximately 10-fold higher than that of the nonhomologous crosses (24). In addition to BMV, homologous RNA recombination has been demonstrated for picornaviruses (18-20, 35), coronaviruses (21, 23, 42), for cowpea chlorotic mottle bromovirus (3), tombusviruses (41), and bacteriophages (31). Homology-driven recombination of non-replicative RNA precursors has been reported for Sindbis virus within the overlapping sequences (34).Homologous crossovers among different BMV RNA segments appear to require common 15-to 60-nucleotide (nt) sequences (26) which are composed of GC-rich regions followed by AU-rich regions (27,28). A proposed templateswitching mechanism (27, 28) predicts that the replicase enzyme pauses (stalls) at the AU-rich sequence on a donor BMV RNA molecule and switches to the acceptor template while the upstream GC-rich region facilitate...
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.
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...
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