SARS Coronavirus Reverse Genetics and Pathogenesis

描述(申请人提供)：冠状病毒是一类有包膜的、单链、阳性的RNA病毒。冠状病毒的基因组是所有RNA病毒中最大的，这使得它们的遗传操作成为一个可怕的问题。我们实验室开发了第一个冠状病毒反向遗传系统，称为靶向RNA重组。该系统已被用来回答有关病毒蛋白结构和功能、宿主物种特异性、病毒粒子组装以及冠状病毒基因组RNA合成异常复杂的机制等基本问题。靶向RNA重组是一种强大而通用的方法，原则上应该适用于所有冠状病毒物种。本应用程序的主要目的是开发并应用该系统来处理引起严重急性呼吸综合征(SARS-CoV)的冠状病毒。为了实现这一点，我们将构建一种跨物种嵌合冠状病毒，它将获得感染小鼠细胞的能力。这种病毒被命名为mSARS-CoV，将成为SARS-CoV反向遗传学基于宿主范围的选择系统的基石。将探索mSARS-CoV和mSARS-CoV突变体的组织培养特性，以深入了解SARS-CoV中八个独特基因的潜在作用，这些基因在其他冠状病毒中没有出现。这些构建的病毒在小鼠宿主中引起的疾病将被描述并与研究得很好的小鼠冠状病毒(MHV)引起的疾病进行比较。此外，该遗传系统将用于回答有关SARS-CoV病毒粒子组装和RNA合成的基本问题，并创建SARS-CoV的小鼠病毒替代品，其病毒粒子蛋白完全来自MHV。预计拟议的工作将产生重要的新信息，作为今后进行更详细研究的基础。它还将产生有价值的工具来测试抗病毒药物，揭示此类药物的靶标，并为疫苗设计提供一种操纵SARS-CoV的手段。

DESCRIPTION (provided by applicant): Coronaviruses are a family of enveloped, single-stranded, positive-sense RNA viruses. The genomes of coronaviruses are the largest among all the RNA viruses, which has made their genetic manipulation a formidable problem. Our laboratory developed the first reverse genetic system for coronaviruses, called targeted RNA recombination. This system has been used to answer fundamental questions about viral protein structure and function, host species specificity, virion assembly, and the unusually complex mechanism of coronavirus genome RNA synthesis. Targeted RNA recombination is a powerful and versatile method that, in principle, should be applicable to all species of coronaviruses. The major object of this application is to develop and apply this system to the coronavirus that is the causative agent for Severe Acute Respiratory Syndrome (SARS-CoV). To accomplish this, we will construct an interspecies chimeric coronavirus that will have gained the ability to infect mouse cells. This virus, designated mSARS-CoV, will serve as the cornerstone for a host-range-based selection system for SARS-CoV reverse genetics. The tissue culture properties of mSARS-CoV and mutants of mSARS-CoV will be explored to gain insights into the potential roles of the eight unique genes in SARS-CoV that do not appear in other coronaviruses. The disease caused by these constructed viruses in the mouse host will be characterized and compared to that caused by the well-studied mouse coronavirus (MHV). Additionally, this genetic system will be used to answer basic questions about the components of SARS-CoV virion assembly and RNA synthesis and to create a mouse virus surrogate of SARS-CoV whose virion proteins are completely derived from MHV. The proposed work is expected to produce significant new information upon which more detailed future studies will be based. It will also generate valuable tools to test antiviral drugs, reveal targets for such drugs, and provide a means to manipulate SARS-CoV for vaccine design.