In vivo and in vitro characterization of an RNA replication enhancer in a satellite RNA associated with Turnip crinkle virus

RNA replication enhancers are cis-acting elements that can stimulate replic ation or transcription of RNA viruses. Turnip crinkle virus (TCV) and satC, a parasitic RNA associated with TCV infections, contain stem-loop structur es that are RNA replication enhancers (P. Nagy, J. Pogany, and A. E. Simon, EMBO J. 1999, 18, 5653-5665). We have found that replacement of 28 nt of t he satC enhancer, termed the motifs-hairpin, with 28 randomized bases reduc ed satC accumulation 8- to 13-fold in Arabidopsis thaliana protoplasts. Del etion of single-stranded flanking sequences at either side of the hairpin a lso affected RNA accumulation with combined alterations at both sides of th e hairpin showing the most detrimental effect in protoplasts. In vitro anal ysis with a partially purified TCV RdRp preparation demonstrated that the m otif1-hairpin in its minus-sense orientation was able to stimulate RNA synt hesis from the satC hairpin promoter (located at the 3' end of plus strands ) by almost twofold. This level of RNA synthesis stimulation is similar to fivefold lower than that observed with a linear promoter, suggesting that a highly stable hairpin promoter is less responsive to the presence of the m otif1-hairpin enhancer than a linear promoter. The motif1-hairpin in its pl us-sense orientation was only 60% as active in enhancing transcription from the hairpin promoter. Since the motif1-hairpin is a hotspot for RNA recomb ination during plus-strand synthesis and since satC promoters located on th e minus-strand are all short linear sequences, these findings support the h ypothesis that the motifs-hairpin is primarily involved in enhancing plus-s trand synthesis. (C) 2001 Academic Press.