Mechanism of RNA Recombination in Carmo- and Tombusviruses: Evidence for Template Switching by the RNA-Dependent RNA Polymerase In Vitro

Abstract

RNA recombination occurs frequently during replication of tombusviruses and carmoviruses, which are related small plus-sense RNA viruses of plants. The most common recombinants generated by these viruses are either defective interfering (DI) RNAs or chimeric satellite RNAs, which are thought to be generated by template switching of the viral RNA-dependent RNA polymerase (RdRp) during the viral replication process. To test if RNA recombination is mediated by the viral RdRp, we used either a purified recombinant RdRp of Turnip crinkle carmovirus or a partially purified RdRp preparation of Cucumber necrosis tombusvirus. We demonstrated that these RdRp preparations generated RNA recombinants in vitro. The RdRp-driven template switching events occurred between either identical templates or two different RNA templates. The template containing a replication enhancer recombined more efficiently than templates containing artificial sequences. We also observed that AU-rich sequences promote recombination more efficiently than GC-rich sequences. Cloning and sequencing of the generated recombinants revealed that the junction sites were located frequently at the ends of the templates (end-to-end template switching). We also found several recombinants that were generated by template switching involving internal positions in the RNA templates. In contrast, RNA ligationbased RNA recombination was not detected in vitro. Demonstration of the ability of carmo-and tombusvirus RdRps to switch RNA templates in vitro supports the copy-choice models of RNA recombination and DI RNA formation for these viruses.Viral RNA recombination, a process that joins together two noncontiguous RNA segments, is an especially powerful tool in virus evolution, since it can rapidly lead to dramatic changes in virus genomes by recombining or rearranging "battle-tested" (i.e., evolutionarily successful) sequences. Accordingly, the significant role of RNA recombination in emergence of new viruses or virus strains is well documented for numerous human, animal, plant, insect, fungal, and bacterial viruses (2, 4, 12, 17, 18, 20, 21, 27-31, 51, 57, 61, 62, 64, 65, 71). In addition to increasing sequence variability, RNA recombination can be an efficient tool for viruses to repair viral genomes, thus contributing to virus fitness (6,14,35,36,53,66). In spite of its significance, our understanding of RNA recombination is incomplete. This is due to the complex nature of RNA recombination and the lack of tractable systems for mechanistic studies.RNA recombination may also play a role in the formation of subviral RNAs that include defective interfering (DI) RNAs associated with many animal and plant viruses. DI RNAs are mainly derived from the parent (helper) virus via sequence deletion(s). The DI RNAs are deficient in replication and/or other functions, which makes them dependent on the helper virus for their survival and spread (50, 70). The best-known DI RNAs among plant viruses are those associated with tombusvirus infections. The tombusvirus DI RNAs are mosai...

Tomato Bushy Stunt Virus Recombination Guided by Introduced MicroRNA Target Sequences

“…The TBSV DI RNAs, derived entirely from the parental viral RNA, are not replication competent alone and depend on the parent virus to replicate them in trans. Recent developments of in vitro systems (19,21) have further enhanced dissection of recombination mechanisms giving rise to TBSV DI RNAs.Of the proposed mechanisms for viral recombination (12, 20), the copy choice or template-switching mechanism is the most widely reported (8,16,18). 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.…”

“…Of the proposed mechanisms for viral recombination (12,20), the copy choice or template-switching mechanism is the most widely reported (8,16,18). 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.…”

The promiscuous evolutionary history of the family Bromoviridae

“…Proportionally, the amount of genetic variability produced after a single recombination event is larger than that produced by single point mutations. Although the importance of recombination was underappreciated in early studies of virus genome evolution, it is now recognized as a widespread phenomenon among positive-strand RNA viruses in animals (Grassly & Holmes, 1997;Holmes et al, 1999;Wilson et al, 1988) and plants (Nagy & Bujarski, 1993;Revers et al, 1996;Aranda et al, 1997;Olsthoorn et al, 2002;Bousalem et al, 2003;Cheng & Nagy, 2003;Moreno et al, 2004;Tan et al, 2004;Bonnet et al, 2005;Urbanowicz et al, 2005;Chare & Holmes, 2006;Weng et al, 2007), as well as in retroviruses such as human immunodeficiency virus type 1 Prljic et al, 2004;Althaus & Bonhoeffer, 2005;Galetto & Negroni, 2005), although it is a rare or even non-existent phenomenon among negativestrand viruses (Chare et al, 2003). There are several mechanisms by which RNA recombination may take place, the most common of which is homologous recombination (Lai, 1992).…”

“…Therefore, the term homologous refers not only to the presence of sequence homology between both parental RNAs but also the necessity of homologous and comparable sites in both parental molecules for the crossovers to take place (Lai, 1992). Aberrant homologous and non-homologous recombinations are less frequent in nature (Lai, 1992).Amongst positive-strand plant RNA viruses, homologous recombination has been reported in vivo and in vitro during mixed infections (Bousalem et al, 2003;Cheng & Nagy, 2003;Moreno et al, 2004;Tan et al, 2004;Bonnet et al, 2005;Urbanowicz et al, 2005;Weng et al, 2007). Focusing on the family Bromoviridae, reports include recombination events in members of the genera Cucumovirus (Férnandez-Cuartero et al, 1994;Fraile et al, 1997;Roossinck et al, 1999;Chen et al, 2002;Bonnet et al, 2005) and Bromovirus (Bujarski & Kaesberg 1986;Allison et al, 1990), as well as between viruses belonging to different genera (de Wispelaere et al, 2005).…”

Sequence characteristics of potato virus Y recombinants

“…They have been found frequently near recombination sites in many viruses, e.g. picornaviruses (Pilipenko et al, 1995), brome mosaic virus (BMV) (Nagy & Bujarski, 1996, 1997Shapka & Nagy, 2004), turnip crinkle virus (TCV) and cucumber necrosis virus (Cheng & Nagy, 2003), citrus tristeza virus (CTV) (Vives et al, 2005), bean pod mottle virus (Zhang et al, 2007), turnip mosaic virus (TuMV) (Ohshima et al, 2007), grapevine fanleaf virus and Arabis mosaic virus (Vigne et al, 2008). On the other hand, RNA secondary structures, especially stemloop structures (hairpins), can promote replicase pausing or act as signals for replicase pausing on the template or nascent RNA (Nagy & Simon, 1997).…”