Mechanism of Viral RNA-dependent RNA Synthesis

RNA病毒需要从RNA模板复制和转录才能引起疾病。这些过程在机理上也是令人感兴趣的，因为病毒复制酶是唯一一类天然地从模板末端起始和终止RNA合成的模板依赖性聚合酶。病毒复制酶（下文称为复制酶）通常由病毒编码的RNA依赖性RNA聚合酶（RdRp）以及另外的病毒和宿主亚基组成。 来自雀麦花叶病毒（BMV）的复制酶可以从感染的植物中富集和溶解，从而允许生物化学表征。 BMV复制酶可以特异性识别BMV RNA，然后通过许多生化步骤，包括从头起始，延伸，终止和RNA重组。这些步骤在体外的要求很好地模仿在体内的复制和重组的要求，从而提供了解最少的模板依赖性聚合酶类的洞察力。本研究的目的是更好地确定BMV复制酶如何识别特定的RNA序列并完成RNA合成的不同步骤，本研究有四个具体目标：1）检测复制酶与核心启动子RNA之间的相互作用; 2）鉴定和表征在RNA合成过程中引起延伸复制酶复合物暂停的RNA序列; 3）检查RNA体外重组的机制;和4）进一步纯化和表征BMV复制酶中的组分。 从执行这些目标中获得的知识将有助于对病毒复制的基本了解。 使用BMV作为模型系统将直接有益于研究较少的系统，这些系统可能导致脑炎、麻疹和肝炎等疾病。 对病毒RNA复制不同步骤的详细了解将为设计高度特异性的抗病毒药物提供靶点。

Replication and transcription from RNA templates are required for RNA viruses to cause diseases. These processes are also mechanistically interesting since the viral replication enzymes are the only class of template-dependent polymerases that naturally initiate and terminate RNA synthesis from the termini of the templates. Viral replication enzymes, hereafter called the replicase, are generally composed of a virally encoded RNA-dependent RNA polymerase (RdRp), and additional viral and host subunits. The replicase from Brome mosaic virus (BMV) can be enriched and solubilized from infected plants, thus permitting biochemical characterization. The BMV replicase can specifically recognize BMV RNAs and then precede through a number of biochemical steps, including de novo initiation, elongation, termination, and RNA recombination. Requirements for these steps in vitro nicely mimic the requirements for replication and recombination in vivo, thus providing insight into the least understood class of template-dependent polymerases. The goal of this project is to better define how the BMV replicase recognizes specific RNA sequences and goes through different steps of RNA synthesis.This research has four specific aims: 1) to examine the interaction between the replicase and core promoter RNAs; 2) to identify and characterize RNA sequences that cause pausing of the elongating replicase complex during RNA synthesis; 3) to examine the mechanism of RNA recombination in vitro; and 4) to further purify and characterize the components in the BMV replicase. Knowledge gained from the execution of these objectives will contribute to the basic understanding of virus replication. The use of BMV as a model system will directly benefit less well-studied systems that can cause diseases such as encephalitis, measles, and hepatitis. The detailed understanding of the different steps in viral RNA replication should uncover targets for the design of highly specific antiviral drugs.