INTRODUCTIONFIG. 1RESULTSFIG. 2DISCUSSIONFIG. 3MATERIALS AND METHODSACKNOWLEDGMENTSREFERENCESIn Vivo and in Vitro Characterization of an RNA Replication Enhancerin a Satellite RNA Associated with Turnip crinkle virusPeter D. Nagy,1,* Judit Pogany,1,* and Anne E. Simon2,†*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 20742Received May 9, 2001; returned to author for revision June 11, 2001; accepted July 20, 2001RNA replication enhancers are cis-acting elements that can stimulate replication or transcription of RNA viruses. Turnipcrinkle virus (TCV) and satC, a parasitic RNA associated with TCV infections, contain stem-loop structures that are RNAreplication enhancers (P. Nagy, J. Pogany, and A. E. Simon, EMBO J. 1999, 18, 5653–5665). We have found that replacementof 28 nt of the satC enhancer, termed the motif1-hairpin, with 28 randomized bases reduced satC accumulation 8- to 13-foldin Arabidopsis thaliana protoplasts. Deletion of single-stranded flanking sequences at either side of the hairpin also affectedRNA 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-senseorientation was able to stimulate RNA synthesis from the satC hairpin promoter (located at the 3⬘ end of plus strands) byalmost 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 alinear promoter. The motif1-hairpin in its plus-sense orientation was only 60% as active in enhancing transcription from thehairpin promoter. Since the motif1-hairpin is a hotspot for RNA recombination during plus-strand synthesis and since satCpromoters located on the minus-strand are all short linear sequences, these findings support the hypothesis that themotif1-hairpin is primarily involved in enhancing plus-strand synthesis. © 2001 Academic PressINTRODUCTIONPlus-strand RNA viruses replicate efficiently in in-fected cells by a two-step process mediated by viralRNA-dependent RNA polymerases (RdRp). First, minus-strand RNAs are synthesized using the plus-strand RNAas template. Second, the new minus strands serve astemplates to produce large quantities of positive-strandRNAs. To recognize and then replicate faithfully only thecognate RNA, the viral RdRp must recognize specificsequences, termed cis-acting elements, which are oftenlocated at the ends of the RNA (de Graaf and Jaspars,1994; Buck, 1996).Cis-acting elements that are absolutely required forviral replication or transcription are called promoters.RNA promoters in most RNA viruses are required forpositioning the polymerase such that initiation of RNAsynthesis de novo, i.e., independent of oligonucleotideprimers, can take place. Replication and transcriptionpromoters have been characterized for many viruses,including bacterial, fungal, animal, and plant viruses (re-viewed by de Graaf and Jaspars, 1994; Buck, 1996).Promoter sequences/structures for these viruses containeither poly(A) tails, pseudoknots, tRNA-like structures,stem-loop structures, or short primary sequences with-out apparent high-order structures. In contrast to promot-ers, RNA replication enhancers are nonessential cis-acting elements that can modulate the level of transcrip-tion and replication in RNA viruses. RNA replicationenhancers have been shown or suggested to play sig-nificant roles in the biology of several RNA viruses (Lai,1998).Turnip crinkle virus (TCV; genus Carmovirus) is one ofthe best characterized model plus-strand RNA virus sys-tems (reviewed by Simon and Nagy, 1996 and in Buck,1996). TCV has a small genome (4054 nt) with two of itsfive genes required for replication. In addition, TCV in-fections are associated with several small parasiticRNAs, such as defective interfering RNAs (Li et al., 1989)and satellite (sat) RNAs (Simon and Howell, 1986). Thesmallest satRNA is designated satD (194 nt). An unusualsatRNA is the recombinant satC (356 nt) with the 5⬘portion derived from satD and the 3⬘ portion originatingfrom two short noncontiguous regions from the 3⬘ regionof TCV genomic RNA (Fig. 1A). Their small size, lack ofopen reading frames, and ability to modulate viral symp-toms make satRNAs excellent models for studies onreplication, recombination, and symptom production byviral RNAs.In vitro and in vivo analyses of sequences that affect1These authors contributed equally to this work.2To whom correspondence and reprint requests should be ad-dressed. Fax: 301-314-7930. E-mail: [email protected] 288, 315–324 (2001)doi:10.1006/viro.2001.1099, available online at http://www.idealibrary.com on0042-6822/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.315minus- and plus-strand synthesis revealed the presenceof at least five elements that are required for or enhanceaccumulation of satC. The plus-strand contains a 3⬘-terminal 29-nt hairpin promoter that is sufficient for com-plementary strand synthesis in vitro (Song and Simon,1995). Site-specific mutagenesis and in vivo genetic se-lection (SELEX) revealed a role for both sequence andstructure of the hairpin in directing minus-strand synthe-sis in vivo (Stupina and Simon, 1997; Carpenter andSimon, 1998). Four elements on minus-strand satC havebeen identified as important for plus-strand synthesis. Atthe 3⬘ end of minus strands is a 6-nt sequence [Carmo-virus Consensus Sequence or CCS (C2–3A/U A/U A/U)]that is conserved among viruses of the genus Carmovi-rus and is required for plus-strand satC synthesis in vivo(Guan et al., 2000a). 5⬘ of this sequence in satC minusstrands (positions 11 to 21) is an 11-nt sequence calledthe 3⬘ proximal element (3⬘ PE) that also contains a CCSand can serve as an independent promoter for comple-mentary-strand synthesis in vitro (Guan et al., 1997,2000a). A second sequence in satC minus strands calledthe 5⬘ proximal element or 5⬘ PE (positions 302 to 315)can also function as an independent promoter in vitro(Guan et al., 1997), is highly sequence specific, and isrequired for plus-strand synthesis in vivo (Guan et al.,2000b).A fourth