Recombination of 5′ subgenomic RNA3a with genomic RNA3 of Brome mosaic bromovirus in vitro and in vivo

RNA–RNA recombination salvages viral RNAs and contributes to their genomic variability. A recombinationally-active subgenomic promoter (sgp) has been mapped in Brome mosaic bromovirus (BMV) RNA3 (Wierzchoslawski et al., 2004. J. Virol.78, 8552–8864) and mRNA-like 5′ sgRNA3a was characterized (Wierzchoslawski et al., 2006. J. Virol. 80, 12357–12366). In this paper we describe sgRNA3a-mediated recombination in both in vitro and in vivo experiments. BMV replicase-directed co-copying of (−) RNA3 with wt sgRNA3a generated RNA3 recombinants in vitro, but it failed to when 3′-truncated sgRNA3a was substituted, demonstrating a role for the 3′ polyA tail. Barley protoplast co-transfections revealed that (i) wt sgRNA3a recombines at the 3′ and the internal sites; (ii) 3′-truncated sgRNA3as recombine more upstream; and (iii) 5′-truncated sgRNA3 recombine at a low rate. In planta co-inoculations confirmed the RNA3–sgRNA3a crossovers. In summary, the non-replicating sgRNA3a recombines with replicating RNA3, most likely via primer extension and/or internal template switching.