Virology 238, 000-000 (1997) Article no. VY978850

Analysis in Vivo of Turnip Crinkle Virus Satellite RNA C Variants with Mutations in the 3'-Terminal Minus-Strand Promoter

Vera Stupina and Anne E. Simon

Department of Biochemistry and Molecular Biology and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003

Turnip crinkle virus and its associated RNA, sat-RNA C, share similar, but not identical hairpins near their 3' ends and terminate with CCUGCCC-OH, which forms a single-stranded tail. With an in vitro transcription system containing partially purified TCV RdRp, the 3'-terminal 29 bases making up the hairpin and single-stranded tail were previously demonstrated to be required for transcription, and alterations in the stem, but not the loop, could affect template activity (C. Song and A. E. Simon, 1995, J. Mol. Biol. 254, 6-14). We have now analyzed sat-RNA C mutants in the 3' hairpin for ability to accumulate in vivo. While active templates in vitro were able to accumulate in vivo, some very weak templates in vitro were also able to accumulate in vivo without reversion or second-site alterations. Computer models of hairpin structure indicated that biologically active promoters could have hairpins less stable than wild type, with loops of variable length and sequence, and without a need for a 6-base single-stranded tail. In addition, transcripts containing compensatory exchanges in the upper stem region that had limited activity in vitro were biologically active in vivo, indicating that positioning of specific bases in the stem is not required to produce an active minus-strand promoter.