Key result
The RNA replication enhancer RIII(-) stimulated plus-strand RNA synthesis by up to 20-fold by binding to the viral replicase and interacting with the core promoter via a long-distance RNA-RNA interaction.
The RNA replication enhancer in tombusviruses stimulates plus-strand RNA synthesis by binding to the viral replicase and interacting with the core promoter via long-distance RNA-RNA interaction.
May inform antiviral strategies targeting RNA enhancers; leaves open extension to human pathogens.
Replication of RNA viruses is regulated by cis-acting RNA elements, including promoters, replication silencers, and replication enhancers (REN). To dissect the function of an REN element involved in plus-strand RNA synthesis, we developed an in vitro trans-replication assay for tombusviruses, which are small plus-strand RNA viruses. In this assay, two RNA strands were tethered together via short complementary regions with the REN present in the nontemplate RNA, whereas the promoter was located in the template RNA. We found that the template activity of the tombusvirus replicase preparation was stimulated in trans by the REN, suggesting that the REN is a functional enhancer when located in the vicinity of the promoter. In addition, this study revealed that the REN has dual function during RNA synthesis. (i) It binds to the viral replicase. (ii) It interacts with the core plus-strand initiation promoter via a long-distance RNA-RNA interaction, which leads to stimulation of initiation of plus-strand RNA synthesis by the replicase in vitro. We also observed that this RNA-RNA interaction increased the in vivo accumulation and competitiveness of defective interfering RNA, a model template. We propose that REN is important for asymmetrical viral RNA replication that leads to more abundant plus-strand RNA progeny than the minus-strand intermediate, a hallmark of replication of plus-strand RNA viruses.
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Panavas et al. (2005) studied Tombusvirus replication. RNA Replication Enhancer (REN) RIII(-) vs. Templates lacking REN or with mutated sequences was evaluated on RNA synthesis from cPR promoter. The RNA replication enhancer RIII(-) stimulated plus-strand RNA synthesis by up to 20-fold by binding to the viral replicase and interacting with the core promoter via a long-distance RNA-RNA interaction.
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