Dual mechanisms for sgRNA3a-mediated recombination with genomic RNA3 of brome mosaic bromovirus: The role of cis-acting motifs and viral coat protein

RNA viruses are nature's swiftest evolvers due to their high recombination frequency. Most of them recombine via the template switch mechanism where RNA polymerase switches mid-replication from one molecule to another using the newly synthesized RNA for priming on the acceptor template.;Brome mosaic bromovirus (BMV) is a representative of the family Bromoviridae with a tripartite RNA genome that supports recombination. The most recombinationally active region was previously detected within the subgenomic promoter (sgp) of RNA3, also shown to participate in the formation of the 5' co-terminal subgenomic (sg) RNA3a.;The multifunctional character of sgp suggested the possible involvement of the sgRNA3a in homologous recombination. This was tested by co-transfection of barley protoplasts with variants of RNA3 and sgRNA3a. The experiments revealed that the full-length sgRNA3a mediates homologous crossovers with genomic RNA3. The reduction in positive-strand RNA3 or its absence at the early stages of infection increased the frequency of crossovers, likely due to reduced competition with RNA3 replication and better access to negative strands. Also, the presence of an unpaired and unstructured 3' sgRNA3a polyA tail was shown to be an essential factor supporting sgRNA3a priming on genomic RNA3. The subsequent experiments with the 3'- and 5'- trucated sgRNA3a derivatives mapped the additional crossover sites within RNA3 to the cis-acting motifs: 5' untranslated region (5'UTR), the packaging element (PE), and the intergenic B-box. Both the B-box and PE were shown to co-operate during sgRNA3a-mediated recombination, likely by their mutual ability to bind the viral structural coat protein (CP). Consequently, the role of the CP in mediating homologous crossovers was addressed. The lack of CP expression and the expression of CP with modified domains recognizing RNA motifs significantly lowered the recombination rate, likely due to disrupted CP/RNA interactions and/or the CP oligomerization.;The results show that the non-replicating ss RNA (sgRNA3a) can recombine with the replicating viral RNA via several mechanisms involving primer extension and template switching mediated by coat protein. The BMV CP should be considered a new protein factor modulating homologous crossovers by binding to the specific RNA motifs and bringing the recombining substrates into proximity.