Evolution of virus-derived sequences for high-level replication of a subviral RNA

Turnip crinkle virus (TCV) and its 356-nt satellite RNA satC share 151 nt of 3′-terminal sequence, which contain 8 positional differences and are predicted to fold into virtually identical structures, including a series of four phylogenetically inferred hairpins. SatC and TCV containing reciprocal exchanges of this region accumulate to only 15% or 1% of wild-type levels, respectively. Step-wise conversion of satC and TCV 3′-terminal sequences into the counterpart's sequence revealed the importance of having the cognate core promoter (Pr), which is composed of a single hairpin that differs in both sequence and stability, and an adjacent short 3′-terminal segment. The negative impact of the more stable TCV Pr on satC could not be attributed to lack of formation of a known tertiary interaction involving the 3′-terminal bases, nor an effect of coat protein, which binds specifically to TCV-like Pr and not the satC Pr. The satC Pr was a substantially better promoter than the TCV Pr when assayed in vitro using purified recombinant TCV RdRp, either in the context of satC or when assayed downstream of non-TCV-related sequence. Poor activity of the TCV Pr in vitro occurred despite solution structure probing indicating that its conformation in the context of satC is similar to the active form of the satC Pr, which is thought to form following a required conformational switch. These results suggest that evolution of satC following its initial formation generated a Pr that can function more efficiently in the absence of additional TCV sequence that may be required for full functionality of the TCV Pr.