RNA elements required for RNA recombination function as replication enhancers in vitro and in vivo in a plus-strand RNA virus

RNA replication requires cis-acting elements to recruit the viral RNA-dependent RNA polymerase (RdRp) and facilitate de novo initiation of complementary strand synthesis. Hairpins that are hot spots for recombination in the genomic RNA of turnip crinkle virus (TCV) and satellite (sat)-RNA C, a parasitic RNA associated with TCV infections, stimulate RNA synthesis 10-fold from a downstream promoter sequence in an in vitro assay using partially purified TCV RdRp. Artificial hairpins had an inhibitory effect on transcription, RNA accumulation in single cells was enhanced 5- to 10-fold when the natural stem-loop structures were inserted into a poorly accumulating sat-RNA. The effect of the stem-loop structures on RNA replication was additive, with insertion of three stem-loop RNA elements increasing sat-RNA accumulation to the greatest extent (25-fold). These stent-loop structures do not influence the stability of the RNAs in vivo, but may serve to recruit the RdRp to the template.