Virology 268, 345-354 (2000)
Analysis of cis-acting sequences involved in plus-strand synthesis of a TCV-associated satellite RNA identifies a new carmovirus replication element
Hancheng Guan, Clifford D. Carpenter and Anne E. Simon
Department of Biochemistry and Molecular Biology and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003
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 carmovirus consensus sequence (CCS) “3’OH-CC1-2 (A/U)(A/U)(A/U)”. Single mutations in the 3’-terminal CCS of satC minus strands 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.