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

Abstract

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...

Risks from GMOs due to Horizontal Gene Transfer

“…Using Cauliflower mosaic virus as a model, over 50% of viral genomes recovered after a single host infection were recombinant with an estimated baseline recombination frequency of 2−4 × 10 −5 per base each replication cycle (Froissart et al, 2005). Other studies support these findings of recombination amongst many groups of viruses (Aaziz and Tepfer, 1999;Banner and Lai, 1991;Bruyère et al, 2000;Revers et al, 1996;Rokyta et al, 2006) and viroids (Keese and Symons, 1985;Rezaian, 1990).…”

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

“…It is known that many viruses undergo recombination during infection (Bujarski, 2013). BMV undergoes recombination during infection and is a model virus for RNA recombination studies (Bujarski and Kaesberg, 1986;Nagy and Bujarski, 1996;Bruyere et al, 2000;Alejska et al, 2005;Urbanowicz et al, 2005;Kwon and Rao, 2012;Kolondam et al, 2015). Our modeling of secondary structures for RNA3 from both vector species (original or modified) with inserts indicated that, regardless of the insert fragment modeled, there was a difference in structure between the original BMVF13m RNA3 vector sequence and the modified BMVCP5 RNA3 vector sequence (Supplemental Figs.…”

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

“…Evidence for the occurrence of the RNA recombination phenomenon either by means of in silico sequence comparison (reviewed by Chare & Holmes, 2006) or by its occurrence in mixed infections (Aaziz & Tepfer, 1999a;de Wispelaere et al, 2005;Meier & Truve, 2006), or under experimental conditions by applying high selection pressure, has been given for numerous plant virus species (Aaziz & Tepfer, 1999b;Simon & Bujarski, 1994). However, knowledge concerning RNA recombination mechanisms and parameters affecting these processes has been obtained mainly from a limited number of positivestrand RNA model virus species, namely brome mosaic virus (BMV) and cowpea chlorotic mottle virus, both belonging to the genus Bromovirus (Alejska et al, 2005;Allison et al, 1996;Bruyere et al, 2000; in supergroup III (van der Heijden & Bol, 2002). Supergroup II RNA recombination model viruses are tomato bushy stunt virus (TBSV) (genus Tombusvirus) (Panavas & Nagy, 2003), turnip crinkle virus (TCV) (genus Carmovirus) (Cascone et al, 1993;Zhang et al, 1991) and the RNA bacteriophage Qb (Brown & Gold, 1996).…”

“…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.…”