In vitro characterization of late steps of RNA recombination in turnip crinkle virus
I: the role of the motif1-hairpin structure

Peter D. Nagy and Anne E. Simon

Department of Biochemistry and Molecular Biology, Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003.

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 et el., 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 motif'-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 wildtype and mutated motif1-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.