In vitro characterization of late steps of RNA recombination in turnip crinkle virus. I. Role of motif1-hairpin structure.

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 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.