RUI: Regulation of the Pyrimidine Pathway In Plants

摘要MCB-0078152。PI：Peter D.纳吉RNA复制是病毒发病机制中的一个中心过程，以前的工作主要集中在启动子元件上。PI和其他人发现了新的顺式作用元件，如上调RNA合成的RNA复制增强子和下调RNA合成的RNA复制沉默子，改变了我们对RNA病毒复制的看法。这些RNA复制增强子和沉默子元件不仅在复制中发挥重要作用，而且还可能影响病毒的适应性和进化以及它们在感染宿主中引起的症状。缺陷干扰RNA（Defective interfering RNA，DI RNA）由于其简单的基因组结构和强大的复制能力，成为研究RNA复制过程中顺式作用元件的重要模型。DI RNA是与许多病毒感染相关的高度竞争性寄生RNA。DI RNA与它们的辅助（亲本）病毒竞争，导致辅助病毒的复制速率降低，并且经常导致宿主中的症状减弱。在本研究中待测试的假设是DI RNA相对于辅助病毒的上级竞争性是由于（i）顺式作用元件如DI RNA中的启动子和复制增强子的有利定位，和/或（ii）DI RNA中可能在DI RNA形成期间已经缺失的一些复制沉默元件的缺失。本研究以植物的模式（+）链RNA病毒-番茄矮病毒（Tombusviruses）为研究对象，利用新近开发的无细胞复制酶系统，对番茄矮病毒复制增强子和复制沉默子进行了表征，并通过检测复制增强子和复制沉默子对番茄矮病毒复制酶蛋白与模板结合的直接影响，阐明了番茄矮病毒复制增强子和复制沉默子对RNA转录的促进和抑制机制。这些研究将使我们更好地理解被复制的RNA上的序列对病毒RNA合成的调控。 这些研究还将阐明某些不完全病毒变异体减弱原始完整病毒感染的能力。这些变异体被称为有缺陷的干扰RNA。 它们来源于感染包括植物在内的多种生物的病毒。

ABSTRACT MCB-0078152. PI: Peter D. Nagy. Previous work on RNA replication, a central process in viral pathogenesis, has focused on promoter elements. The discovery by the PI and others of novel cis-acting elements, such as RNA replication enhancers that up-regulate RNA synthesis and RNA replication silencers that down-regulate RNA synthesis, changes our view of RNA virus replication. These RNA replication enhancer and silencer elements may play major roles not only in replication, but they may also affect the fitness and evolution of viruses and the symptoms they cause in infected hosts. Defective interfering (DI) RNAs, due to their simple genomes and robust replication are attractive model systems for understanding the role of cis-acting elements in RNA replication. DI RNAs are highly competitive, parasitic RNAs associated with many virus infections. DI RNAs compete against their helper (parental) viruses, resulting in a decreased replication rate of the helper virus and frequently leading to symptom attenuation in the host. The hypothesis to be tested in this research is that the superior competitiveness of the DI RNAs relative to the helper virus is due to (i) favorable positioning of cis-acting elements, such as promoters and replication enhancers in DI RNAs, and/or (ii) the absence of some replication silencer elements in DI RNAs that may have been deleted during DI RNA formation. This research uses tombusviruses, model (+)-strand RNA viruses of plants, to: (i) characterize replication enhancer and silencer elements by using a recently developed cell-free replicase system; (ii) elucidate the mechanism of enhancer-mediated stimulation and silencer-mediated inhibition of RNA transcription through testing the direct effect of RNA enhancer and silencer elements on template binding by tombusvirus replicase proteins expressed in E. coli.These studies will lead to a better understanding of regulation of viral RNA synthesis by sequences on the RNA being copied. The studies will also elucidate the ability of certain incomplete viral variants to attenuate infections by the original, complete viruses from which they were derived. These variants are called defective interfering RNA's. They are derived from viruses that infect a wide range of organisms, including plants.