Analysis of cis-acting sequences involved in plus-strand synthesis of a turnip crinkle virus-associated satellite RNA identifies a new carmovirus replication element.

Satellite RNA C (satC) is a 356-base subviral RNA associated with turnip crinkle virus (TCV). A 3'-proximal element (3'-UCCCAAAGUAU) located 11 bases from the 3' terminus of satC minus strands can function as an independent promoter in an in vitro RNA-dependent RNA polymerase (RdRp) transcription system. Furthermore, in the absence of a 5'-proximal element, the 3'-proximal element is required for complementary strand synthesis in vitro. Site-directed mutagenesis was conducted to investigate the functional significance of this element and the 3' minus-strand terminal sequence "3'-OH-CCCUAU," which contains the minus-strand 3'-end sequence "3'-OH-CC(1-2)(A/U)(A/U)(A/U)" found in all carmovirus RNAs. Single mutations in the 3'-terminal sequence, which we have named the carmovirus consensus sequence (CCS), suppressed satC plus-strand synthesis to undetectable levels in protoplasts while still permitting some minus-strand synthesis. However, single and multiple mutations introduced into the 3'-proximal element had little or no effect on satC accumulation in protoplasts. In vivo genetic selection (SELEX) of the minus-strand 3'-terminal 21 bases revealed that all satC species accumulating in plants contained the 3' CCS. In addition, the 3'-proximal element preferentially contained a sequence similar to the CCS and/or polypurines, suggesting that this element may also contribute to accumulation of satC in vivo.