Importance of sequence and structural elements within a viral replication repressor

Efficient replication of plus-strand RNA viruses requires a 3′ proximal core promoter and an increasingly diverse inventory of supporting elements such as enhancers, repressors, and 5′ terminal sequences. While core promoters have been well characterized, much less is known about structure-functional relationships of these supporting elements. Members of the genus Carmovirus family Tombusviridae contain a hairpin (H5) proximal to the core promoter that functions as a repressor of minus-strand synthesis in vitro through an interaction between its large symmetrical internal loop (LSL) and 3′ terminal bases. Turnip crinkle virus satellite RNA satC with the H5 of carmovirus Japanese iris necrosis virus or Cardamine chlorotic fleck virus (CCFV) did not accumulate to detectable levels even though 3′ end base-pairing would be maintained. Replacement of portions of the satC H5 with analogous portions from CCFV revealed that the cognate LSL and lower stem were of greater importance for satC accumulation than the upper stem. In vivo selex of the H5 upper stem and terminal GNRA tetraloop revealed considerable plasticity in the upper stem, including the presence of three- to six-base terminal loops, allowed for H5 function. In vivo selex of the lower stem revealed that both a stable stem and specific base pairs contributed to satC fitness. Surprisingly, mutations in H5 had a disproportionate effect on plus-strand accumulation that was unrelated to the stability of the mutant plus-strands. In addition, fitness to accumulate in plants did not always correlate with enhanced ability to accumulate in protoplasts, suggesting that H5 may be multifunctional.