MECHANISM OF TRANSCRIPTION OF CORONAVIRUS

本申请的总体目标是了解 小鼠肝炎病毒（MHV-JHM）RNA合成机制， 鼠冠状病毒冠状病毒是RNA的一大家族 引起呼吸道、胃肠道和神经系统疾病的病毒 包括人类在内的各种动物的疾病。 状病毒 RNA合成被认为是通过一种不连续的机制发生的 这一点还没有完全理解。 冠状病毒也经历 高频率的不连续拷贝选择RNA重组。 为了理解这些不寻常的不连续机制， 冠状病毒RNA合成，我们将分析负责的酶 对于病毒RNA的合成，依赖RNA的RNA聚合酶。的 冠状病毒RNA聚合酶是作为一个大的前体合成的 必须加工成功能亚基的多蛋白。 我们 假设一系列蛋白水解加工事件调节 冠状病毒RNA聚合酶的活性和特异性。 我们 建议通过以下方法鉴定聚合酶的加工亚基： 产生针对RNA聚合酶的特异性结构域的抗体， 并利用这些抗体来识别病毒蛋白质， 细胞 为了确定RNA聚合酶多蛋白是如何 加工成亚基，我们就产生了聚合酶多蛋白 无细胞系统中的产物和监测翻译产物 蛋白水解活性。 识别组件的功能 RNA聚合酶多蛋白，我们正在体外开发 活性测定和鉴定导致 温度敏感突变体中RNA合成的损失 冠状病毒。 我们还将确定是否需要主机因素 通过测试冠状病毒复制来确定RNA合成或RNA稳定性 当宿主因子被抑制或不存在时。 总的来说，这些研究 旨在识别冠状病毒RNA的功能域- 依赖性RNA聚合酶。 如果我们能理解 冠状病毒RNA依赖的RNA聚合酶的产生，以及如何 它转录并复制病毒的遗传信息， 设计特异性药物阻断病毒复制和致病性。

The overall objective of this application is to understand the mechanism of RNA synthesis of mouse hepatitis virus (MHV-JHM), a murine coronavirus. Coronaviruses are a large family of RNA viruses which cause respiratory, gastrointestinal and neurological diseases in a variety of animals including humans. Coronavirus RNA synthesis is proposed to occur by a discontinuous mechanism which is not fully understood. Coronaviruses also undergo discontinuous copy-choice RNA recombination at a high frequency. To understand these unusual discontinuous mechanisms of coronavirus RNA synthesis, we will analyze the enzyme responsible for viral RNA synthesis, the RNA -dependent RNA polymerase. The coronavirus RNA polymerase is synthesized as a large precursor polyprotein which must be processed into functional subunits. We hypothesize that a cascade of proteolytic processing events regulates the activity and specificity of the coronavirus RNA polymerase. We propose to identify the processed subunits of the polymerase by generating antibodies to specific domains of the RNA polymerase, and using those antibodies to identify viral proteins from infected cells. To determine how the RNA polymerase polyprotein is processed into subunits, we are generating polymerase polyprotein products in a cell-free system and monitoring the translation products for proteolytic activity. The identify the function of the components of the RNA polymerase polyprotein, we are developing in vitro assays for activity and identifying specific mutations which result in the loss of RNA synthesis in temperature sensitive mutants of coronavirus. We will also determine if host factors are required for RNA synthesis or RNA stability by testing coronavirus replication when host factors are inhibited or absent. Overall, these studies are designed to identify functional domains of the coronavirus RNA- dependant RNA polymerase. If we can understand how the coronavirus RNA -dependent RNA polymerase is generated, and how it transcribes and replicates the viral genetic information, we can design specific drugs to block viral replication and pathogenicity.