In vitro characterization of late steps of RNA recombination in turnip crinkle virus: I. Role of the Motif-Hairpin structure

Peter D. Nagy, Anne E. Simon

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33 Scopus citations

Abstract

Molecular mechanisms of RNA recombination were studied in turnip crinkle carmovirus (TCV), which has a uniquely high recombination frequency and nonrandom crossover site distribution among the recombining TCV-associated satellite RNAs. An in vitro system has been developed that includes a partially purified TCV replicase preparation (RdRp) and chimeric RNAs that resemble the putative in vivo recombination intermediates (Nagy, P. D., Zhang, C., and Simon, A. E. EMBO J. 17, 2392-2403, 1998). This system generates 3'-terminal extension products, which are analogous to the recombination end products. Efficient generation of 3'-terminal extension products depends on the presence of a hairpin structure (termed the motif1- hairpin) that possibly binds to the RdRp. Replacement of the motif1-hairpin with two separate randomized sequences resulted in a basal level of 3'- terminal extension. By using three separate constructs, each carrying similar mutations in the motif1-hairpin, we demonstrate that the role of the motif1- hairpin in 3'-terminal extension is complex and its function is influenced by flanking sequences. In addition to the mutagenesis approach, competition experiments between wild-type and mutated motif 1-hairpin constructs suggest that the TCV RdRp likely recognizes the secondary and/or tertiary structure of the motif1-hairpin, while individual nucleotides play a less important role. Overall, the data shed new light into the mechanism of 3'-terminal extension by a viral RdRp that is analogous to the late steps of RNA recombination in TCV.

Original languageEnglish
Pages (from-to)379-392
Number of pages14
JournalVirology
Volume249
Issue number2
DOIs
StatePublished - Sep 30 1998

Bibliographical note

Funding Information:
We thank Dr. Judit Pogany for critical reading of the manuscript. This work was supported by National Science Foundation Grants MCB-9630191 and MCB-9728277 to A.E.S.

Keywords

  • Plant virus
  • Recombination
  • Replication
  • Satellite RNA
  • Template switching

ASJC Scopus subject areas

  • Virology

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