Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase.
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.
Panavas
T
T
Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546, USA.
Pogany
J
J
Nagy
P D
PD
D018553:Cucumis sativus / Q000821:virology*; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D014026:Nicotiana; D017864:Tombusvirus / Q000201:enzymology / Q000235:genetics*
D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase