Key result
Deletion of satC 3'-terminal bases or alteration of interacting bases enhanced transcription in vitro, indicating that hairpin 5 functions to suppress synthesis of minus strands by sequestering the 3' terminus from the RNA-dependent RNA polymerase.
The study identifies a mechanism for controlling minus-strand synthesis in carmoviruses via a transcriptional repressor hairpin (H5) that sequesters the 3' terminus from the RNA-dependent RNA polymerase.
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Hairpin 5 suppression of minus-strand synthesis leaves open conserved regulation in human plus-strand RNA viruses; hypothesis-generating for antiviral RNA targeting.
Plus-strand viral RNAs contain sequences and structural elements that allow cognate RNA-dependent RNA polymerases (RdRp) to correctly initiate and transcribe asymmetric levels of plus and minus strands during RNA replication. cis-acting sequences involved in minus-strand synthesis, including promoters, enhancers, and, recently, transcriptional repressors (J. Pogany, M. R. Fabian, K. A. White, and P. D. Nagy, EMBO J. 22:5602-5611, 2003), have been identified for many viruses. A second example of a transcriptional repressor has been discovered in satC, a replicon associated with turnip crinkle virus. satC hairpin 5 (H5), located proximal to the core hairpin promoter, contains a large symmetrical internal loop (LSL) with sequence complementary to 3'-terminal bases. Deletion of satC 3'-terminal bases or alteration of the putative interacting bases enhanced transcription in vitro, while compensatory exchanges between the LSL and 3' end restored near-normal transcription. Solution structure analysis indicated that substantial alteration of the satC H5 region occurs when the three 3'-terminal cytidylates are deleted. These results indicate that H5 functions to suppress synthesis of minus strands by sequestering the 3' terminus from the RdRp. Alteration of a second sequence strongly repressed transcription in vitro and accumulation in vivo, suggesting that this sequence may function as a derepressor to free the 3' end from interaction with H5. Hairpins with similar sequence and/or structural features that contain sequence complementary to 3'-terminal bases, as well as sequences that could function as derepressors, are located in similar regions in other carmoviruses, suggesting a general mechanism for controlling minus-strand synthesis in the genus.
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Zhang et al. (2004) studied Turnip crinkle virus (TCV) replication. Mutations in satC RNA (e.g., deletion of 3'-terminal cytidylates) vs. Wild-type satC RNA was evaluated on Minus-strand RNA synthesis in vitro. Deletion of satC 3'-terminal bases or alteration of interacting bases enhanced transcription in vitro, indicating that hairpin 5 functions to suppress synthesis of minus strands by sequestering the 3' terminus from the RNA-dependent RNA polymerase.
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