STRUCTURE AND FUNCTION OF THE CORONAVIRUS REPLICASE

描述：冠状病毒是RNA病毒的一个家族， 胃肠道和神经系统疾病，包括 人类冠状病毒RNA合成是不寻常的，因为病毒mRNA的产生 在此期间，前导RNA和mRNA体序列 成为连续的。此外，复制病毒RNA经历高频率的 RNA重组为了了解病毒RNA的这些独特机制 合成，我们正在表征冠状病毒复制酶， 负责RNA合成和重组。冠状病毒复制酶是 合成为800 kDa多蛋白，由病毒蛋白酶加工。 在这个提议中，我们将研究复制酶多蛋白是如何 在细胞内膜上加工、组装，并在病毒RNA中发挥作用 合成.我们假设某些复制酶产物作为支架 用于将复制酶复合物组装到膜上，并且该组装是 对于功能性复制酶的产生至关重要。使用一系列 针对单个复制酶产物产生的多克隆抗血清，我们将 通过共聚焦和共聚焦显微镜测定复制酶产物的亚细胞定位， 免疫电镜生物化学方法和系统， 复制酶产物的细胞质表达将用于确定 单独的复制酶产物作为完整的膜蛋白，或者如果它们的 本地化依赖于其他复制产品。我们还将使用 细胞质表达系统，以反式表达复制酶产物， 确定它们是否可以补充温度敏感突变体， 病毒RNA合成。这些研究将提供新的信息， 介导不连续mRNA合成和RNA重组的复制酶， 这些事件有助于病毒快速进化，逃避 并阻碍疫苗开发。

DESCRIPTION: Coronaviruses are a family of RNA viruses that cause respiratory, gastrointestinal and neurological diseases in a variety of animals including humans. Coronavirus RNA synthesis is unusual because viral mRNAs are generated via a discontinuous mechanism during which a leader RNA and mRNA body sequence become contiguous. Furthermore, replicating viral RNAs undergo high frequency RNA recombination. To understand these unique mechanisms of viral RNA synthesis, we are characterizing the coronavirus replicase, the enzyme responsible for RNA synthesis and recombination. The corona virus replicase is synthesized as an 800 kDa polyprotein that is processed by viral proteinases. In this proposal, we will investigate how the replicase polyprotein is processed, assembled on intracellular membranes, and functions in viral RNA synthesis. We hypothesize that certain replicase products act as the scaffold for assembly of the replicase complex onto membranes, and that this assembly is essential for the generation of the functional replicase. Using a series of polyclonal antisera generated to individual replicase products, we will determine the subcellular localization of replicase products by confocal and immuno-electron microscopy. Biochemical methods and a system for the cytoplasmic expression of replicase products will be used to determine if individual replicase products act as integral membrane proteins or if their localization is dependent of other replication products. We will also use the cytoplasmic expression system to express replicase products in trans and determine if they can complement temperature sensitive mutants defective in viral RNA synthesis. These studies will provide new information of a viral replicase that mediates discontinuous mRNA synthesis and RNA recombination, events that contribute to the ability of viruses to rapidly evolve, evade the immune system and hamper vaccine development.