Molecular Studies of Genetic RNA–RNA Recombination in Brome Mosaic 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.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...

Section: Methodsmentioning

confidence: 99%

Frequent Homologous Recombination Events between Molecules of One RNA Component in a Multipartite RNA Virus

Bruyere

Wantroba

Flasiński

et al. 2000

“…This occurs when the viral replicase and its attached nascent polynucleotide chain switches viral RNA templates (or jumps locations on the same template) when making cRNA. Some properties for preferred donor/acceptor sites (sequences in the RNA molecule at which viral replicase switches from one template to another) are known for various RNA viruses (3,20,27), suggesting that recombination is not entirely random.…”

mentioning

confidence: 99%

Tomato Bushy Stunt Virus Recombination Guided by Introduced MicroRNA Target Sequences

Kumar

Uratsu

Dandekar

et al. 2009

Previously we described Tomato bushy stunt virus (TBSV) vectors, which retained their capsid protein gene and were engineered with magnesium chelatase (ChlH) and phytoene desaturase (PDS) gene sequences from Nicotiana benthamiana. Upon plant infection, these vectors eventually lost the inserted sequences, presumably as a result of recombination. Here, we modified the same vectors to also contain the plant miR171 or miR159 target sequences immediately 3 of the silencing inserts. We inoculated N. benthamiana plants and sequenced recombinant RNAs recovered from noninoculated upper leaves. We found that while some of the recombinant RNAs retained the microRNA (miRNA) target sites, most retained only the 3 10 and 13 nucleotides of the two original plant miRNA target sequences, indicating in planta miRNA-guided RNA-induced silencing complex cleavage of the recombinant TBSV RNAs. In addition, recovered RNAs also contained various fragments of the original sequence (ChlH and PDS) upstream of the miRNA cleavage site, suggesting that the 3 portion of the miRNA-cleaved TBSV RNAs served as a template for negative-strand RNA synthesis by the TBSV RNA-dependent RNA polymerase (RdRp), followed by template switching by the RdRp and continued RNA synthesis resulting in loss of nonessential nucleotides.Several plant viruses have been developed as tools for various biotechnology applications, including expression platforms for protein production in plants (1, 2, 6) and as gene silencing systems as part of reverse genetics approaches toward understanding host plant gene function (4, 5). For both of these applications, nonviral sequences conferring the desired function are cloned into the virus genome in order to be expressed during replication in plants. One advantage of using viruses engineered with nonviral sequences is flexibility in manipulating these "extra" sequences, which are not essential for viral replication or movement (1). However, recombinant viruses also tend to lose these sequences, causing instability at the insertion site and resulting in loss of function of the recombinant viral vector. The relatively high error rates of viral replicases (7,14,24) and the propensity for recombination events (9) contribute to the instability often seen with some viral vector systems.Recombination events in RNA viruses typically result in joining of two noncontiguous RNA segments (16). These could be sequences from two separate RNA molecules or distant regions of the same molecule. Retention by viruses of favorable sequences is selection driven and eliminates sequences that are unnecessary or negatively affect fitness (11, 31), hence making recombination critical to virus evolution (13, 29). Although phylogenetic analyses predict that recombination events have affected evolution for essentially all groups of RNA viruses (3), some viruses appear to be more prone to recombination than others. For example, plantinfecting supergroup II viruses of the family Tombusviridae appear to undergo frequent recombination, as is supported by the ...

“…The 3Ј noncoding regions (3Ј NCR) of the three BMV RNAs share high sequence identity, and homologous recombination occurs frequently among these regions (7,8). In particular, the so-called region R sequence, which is part of the wild-type (wt) 3Ј NCRs of RNA2 and -3, can support homologous crossovers between RNA2 and RNA3 (32).…”

mentioning

confidence: 99%

RNA Recombination in Brome Mosaic Virus: Effects of Strand-Specific Stem-Loop Inserts

Olsthoorn

Bruyere

Dzianott

et al. 2002

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 negativestrand synthesis.RNA recombination is a general phenomenon in animal, plant, and bacterial RNA viruses, and it plays an important role in the fitness and evolution of virus genomes (24,28,36). Model systems to study RNA recombination have been developed for the Brome mosaic bromovirus (BMV), coronaviruses, poliovirus, Turnip crinkle carmovirus (TCV), tombusviruses, and for RNA phage Q␤ (5, 6). Most of the RNA recombination events are thought to occur via a copy-choice mechanism. The evidence for participation of the replicase (RdRp) proteins in recombination has been provided for BMV (15)(16)(17)35). Other proposed mechanisms are cleavage/religation (25,28,45) and transesterification (10).According to a copy-choice model, prior to the actual switch the replicase stalls or dislodges from its template (21,28,36), which may occur at the 5Ј end of the template (47, 49), at a template break (39), or at the nascent strand stem-loop structure. The latter is analogous to rho-independent transcription termination (46, 50), although it may be distinct from a dislodging RdRp in a copy-choice model. Polymerase stalling may be induced by RNA secondary and/or tertiary structures, by homopolymer runs, or by the misincorporation of nucleotides (12,20,21,48,50).The success of the switch to the acceptor template can be influenced by a number of factors (5,6,23,26,28), such as (i) intermolecular RNA-RNA interactions (18, 31), (ii) intramolecular RNA-RNA interactions, or (iii) RNA-protein interactions (9, 36, 38). Altogether, RNA recombination appears to be a multistep process that involves primary and secondary structures, and the progeny recombinants are further selected on the basis of their fitness (43).The intersegmental...