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In Vivo and in Vitro Characterization of an RNA Replication Enhancer



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Virology 288 315 324 2001 doi 10 1006 viro 2001 1099 available online at http www idealibrary com on In Vivo and in Vitro Characterization of an RNA Replication Enhancer in a Satellite RNA Associated with Turnip crinkle virus Peter D Nagy 1 Judit Pogany 1 and Anne E Simon 2 Department of Plant Pathology University of Kentucky Lexington Kentucky 40546 and Department of Cell Biology and Molecular Genetics University of Maryland College Park College Park Maryland 20742 Received May 9 2001 returned to author for revision June 11 2001 accepted July 20 2001 RNA replication enhancers are cis acting elements that can stimulate replication or transcription of RNA viruses Turnip crinkle virus TCV and satC a parasitic RNA associated with TCV infections contain stem loop structures 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 the satC enhancer termed the motif1 hairpin with 28 randomized bases reduced satC accumulation 8 to 13 fold in Arabidopsis thaliana protoplasts Deletion of single stranded flanking sequences at either side of the hairpin also affected RNA accumulation with combined alterations at both sides of the hairpin showing the most detrimental effect in protoplasts In vitro analysis with a partially purified TCV RdRp preparation demonstrated that the motif1 hairpin in its minus sense orientation was able to stimulate RNA synthesis from the satC hairpin promoter located at the 3 end of plus strands by almost twofold This level of RNA synthesis stimulation is fivefold lower than that observed with a linear promoter suggesting that a highly stable hairpin promoter is less responsive to the presence of the motif1 hairpin enhancer than a linear promoter The motif1 hairpin in its plus sense orientation was only 60 as active in enhancing transcription from the hairpin promoter Since the motif1 hairpin is a hotspot for RNA recombination during plus strand synthesis and since satC



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