Virology 268, 355-363 (2000)

Requirement of a 5’-proximal linear sequence on minus strands for plus-strand synthesis of a satellite RNA associated with TCV

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

Viral RNA replication begins with specific recognition of cis-acting RNA elements by the viral RNA-dependent RNA polymerase (RdRp) and/or associated host factors. A short RNA element (3’-AACCCCUGGGAGGC) located 41 bases from the 5’ end of minus strands of satC, a subviral RNA naturally associated with turnip crinkle virus (TCV), was previously identified as important for plus-strand synthesis using an in vitro RdRp assay (Guan H, Song C, Simon AE, 1997, RNA 3:1401-1412). To examine the functional significance of this element in RNA replication, mutations were introduced into the consecutive C residues in the element. A single mutation of the 3’ C residue resulted in undetectable levels of satC plus strands when transcripts were assayed in protoplasts and suppressed transcription directed by the element in vitro. Synthesis of plus strands was inhibited more substantially than that of minus strands in protoplasts suggesting that this 5’-proximal RNA element is required for satC plus-strand synthesis. In addition, in vivo genetic selection revealed a strict requirement for 10 of the 14 nucleotides of the element, indicating that the primary sequence is essential for RNA accumulation.