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

Abstract

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...

Evidence for similarity-assisted recombination and predicted stem-loop structure determinant in potato virus X RNA recombination

“…These double-stranded structures might lead to RdRp stalling or template dissociation (Jager & Pata, 1999;Nagy & Simon, 1997) and possibly direct the template switch to the nonstructured loop. This finding, however, is in contrast to the homologous recombination results obtained with BMV (Olsthoorn et al, 2002), where a reduction in recombinant accumulation and no evidence for enhanced RdRp pausing and detachment were detected when stem-loop structures were introduced into the parental sequences. On the other hand, it has been demonstrated for BMV that intercistronic subgenomic CP promoter (sgPr) sequences, consisting of an AU-rich region and an RdRp-binding stem-loop structure, increase homologous recombination activity (Bruyere et al, 2000;Nagy et al, 1999;Wierzchoslawski et al, 2004).…”

“…Several variants of nascent-strand transfer are conceivable (Gmyl & Agol, 2005;Nagy & Simon, 1997), but experimental evidence is still lacking. Important structural factors of the template or nascent strand that are used for classification have been identified in these model viruses and can be subdivided into primary factors, such as sequence similarity, AU-rich motifs, sequence complementarity and replicative cis elements, and secondary structures, such as stem-loops (Bruyere et al, 2000;Nagy & Bujarski, 1993Olsthoorn et al, 2002;Shapka & Nagy, 2004). So far, nonreplicative RNA recombination, possibly based on RNA breakage and ligation, has only been detected in bacteriophage Qb and polioviruses (Chetverin et al, 1997;Chetverina et al, 1999;Gallei et al, 2004;Gmyl et al, 1999Gmyl et al, , 2003, but not yet in plant viruses.…”

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS

“…It is generally considered that most RNA recombination events proceed through template switching and that RNA structures that cause the polymerase to pause may enhance its release from the donor template strand, leading to association with the acceptor template strand (Sztuba-Soli nska et al, 2011). Olsthoorn et al (2002) determined that insertion of structured stem-loop sequences into regions where recombination occurred could influence the accumulation of recombinants. It will be important to determine whether structural differences caused by the nucleotide deletions and substitutions in our modified vector altered stem loops in planta, leading to decreased recombination by this vector.…”

Genetic characterization of Pepino mosaic virus isolates from Belgian greenhouse tomatoes reveals genetic recombination

“…However, some regions, so-called recombination hot spots, appear to display a higher recombination frequency which is generally explained by specific secondary structures such as stem loops ******************** * ***** * *** **** ** *************** ** ** ** ** **** * **** **** **** *************** * ******** ************************* Fig. 3 (continued) (Olsthoorn et al 2002). The occurrence of such recombination hot spots could not be demonstrated based on our data, as multiple recombination regions were identified for the sequences obtained in our study.…”