Mechanism of Defective Interfering RNA Replication and Interference with Helper Virus Infections

摘要：MCB-0078152。派：彼得·D·纳吉。RNA复制是病毒致病过程中的一个核心过程，以前的工作主要集中在启动子元件上。PI和其他新的顺式作用元件的发现，如上调RNA合成的RNA复制增强剂和下调RNA合成的RNA复制抑制剂，改变了我们对RNA病毒复制的看法。这些RNA复制增强子和沉默元件不仅可能在复制过程中发挥重要作用，而且还可能影响病毒的适合性和进化以及它们在受感染宿主中引起的症状。缺陷干扰(DI)RNA由于其简单的基因组和强大的复制能力，是理解顺式作用元件在RNA复制中作用的有吸引力的模型系统。Di RNA是一种高度竞争的寄生RNA，与许多病毒感染有关。Di RNA与它们的辅助(亲本)病毒竞争，导致辅助病毒的复制率降低，并经常导致宿主的症状减弱。本研究要检验的假设是，DI RNAs相对于辅助病毒的优势竞争力是由于(I)顺式作用元件的有利位置，例如DI RNAs中的启动子和复制增强剂，和/或(Ii)DI RNAs中没有一些可能在DI RNA形成过程中被删除的复制沉默元件。本研究利用植物模型(+)链RNA病毒：(I)利用最近发展的无细胞复制酶系统对复制增强子和沉默元件进行表征；(Ii)通过检测RNA增强元件和沉默元件对大肠杆菌中表达的番茄病毒复制酶蛋白与模板结合的直接影响，阐明增强因子和沉默元件对RNA转录的刺激和抑制机制。这些研究还将阐明某些不完全病毒变体的能力，以减轻来自它们的原始、完整病毒的感染。这些变异体被称为缺陷干扰RNA。它们来自于感染包括植物在内的各种生物体的病毒。

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.