VIROLOGY 249, 393 - 405 (1998) ARTICLE NO. VY989342

In vitro characterization of late steps of RNA recombination in turnip crinkle virus
II: the role of the priming stem and flanking sequences

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.

Turnip crinkle carmovirus (TCV) has a uniquely hign 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 at., 1998). This system mimics the strand transfer and primer extension steps of recombination events. We characterize in detail three RNA factors that, in addition to the previously characterized motif1-hairpin, can influence the efficient generation of 3'- terminal extension products: (i) a primer binding region, termed the priming stem; (ii) a spacer region; and (iii) a U-rich sequence located 5' of the motif1-hairpin. The priming stem is formed between the acceptor RNA and the nascent RNA synthesized from the donor RNA template in the recombinants. The stability and location of the priming stem relative to the motif1-hairpin, can influence both the efficiency and initiation site of 3'-terminal extension. A short flexible spacer region between the motif1-hairpin and the priming stem can increase the efficiency of 3'-terminal extensions. A U-rich sequence 5' of the motif1-hairpin facilitates 3'-terminal extensions and its function partly overlaps with that of the spacer region. These RNA factors may also affect the late steps of RNA recombination in TCV.