Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase

T. Panavas, J. Pogany, P. D. Nagy

Research output: Contribution to journalArticlepeer-review

71 Scopus citations

Abstract

Tombusviruses are small, plus-sense, single-stranded RNA viruses of plants. A partially purified RNA-dependent RNA polymerase (RdRp) preparation of Cucumber necrosis virus (CNV), which is capable of de novo initiation of complementary RNA synthesis from either plus-strand or minus-strand templates, was used to dissect minimal promoter sequences for tombusviruses and their defective interfering (DI) RNAs. In vitro RdRp assay revealed that the core plus-strand initiation promoter included only the 3′-terminal 11 nucleotides. A hypothetical promoter-like sequence, which has been termed consensus sequence by Wu and White (1998, J. Virol. 72, 9897-9905), is recognized less efficiently by the CNV RdRp than the core plus-strand initiation promoter. The CNV RdRp can efficiently recognize the core plus-strand initiation promoter for a satellite RNA associated with the distantly related Turnip crinkle virus, while artificial AU- or GC-rich 3′-terminal sequences make poor templates in the in vitro assays. Comparison of the "strength" of minimal plus-strand and minus-strand initiation promoters reveals that the latter is almost twice as efficient in promoting complementary RNA synthesis. Template competition experiments, however, suggest that the minimal plus-strand initiation promoter makes an RNA template more competitive than the minimal minus-strand initiation promoter. Taken together, these results demonstrate that promoter recognition by the tombusvirus RdRp requires only short sequences present at the 3′ end of templates.

Original languageEnglish
Pages (from-to)263-274
Number of pages12
JournalVirology
Volume296
Issue number2
DOIs
StatePublished - 2002

Bibliographical note

Funding Information:
We thank Drs. Tom Pirone and Andy White for critical reading of the manuscript and for very helpful suggestions. This work was supported by NSF (MCB0078152) and by the University of Kentucky. This study is Publication No. 01-12-56 of the Kentucky Agricultural Experiment Station.

Keywords

  • Internal initiation
  • Plant virus
  • Plus-strand synthesis
  • RNA promoter
  • Replication
  • Template competition
  • de novo initiation

ASJC Scopus subject areas

  • Virology

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