Requirement of a 5 '-proximal linear sequence on minus strands for plus-strand synthesis of a satellite RNA associated with turnip crinkle virus

Viral RNA replication begins with specific recognition of cis-acting RNA el ements by the viral RNA-dependent RNA polymerase (RdRp) and/or associated h ost factors. A short RNA element (3'-AACCCCUGGGAGGC) located 41 bases from the 5' end of minus strands of satellite RNA C (satC), a 356-base subviral RNA naturally associated with turnip crinkle virus (TCV), was previously id entified as important for plus-strand synthesis using an in vitro RdRp assa y (H. Guan, C. Song, A. E. Simon, 1997, RNA 3, 1401-1412). To examine the f unctional significance of this element in RNA replication, mutations were i ntroduced into the consecutive C residues in the element. A single mutation of the 3'-most C residue resulted in undetectable levels of satC plus stra nds when transcripts were assayed in protoplasts and suppressed transcripti on directed by the element in vitro. However, satC minus strands were detec table at 6 h postinoculation (hpi) of protoplasts, accumulating to about 10 % of wild-type levels at 24 hpi. This mutation, when in the plus-sense orie ntation, had little or no effect on minus-strand synthesis from full-length satC plus strands in vitro, suggesting that the 5'-proximal RNA element is required for satC plus-strand synthesis. in addition, in vivo genetic sele ction revealed a strict requirement for 10 of the 14 nucleotides of the ele ment, indicating that the primary sequence is essential for RNA accumulatio n, (C) 2000 Academic Press.