SARS-CoV reverse genetics: expression of small ORFs

描述（由申请人提供）： 在过去的一年里，一种新的冠状病毒（SARS-CoV）已被确定为严重急性呼吸综合征（SARS）的病原体。鉴于死亡率高，缺乏有效的治疗，以及这种疾病何时可能复发和复发频率的不确定性，SARS疫苗和抗病毒药物的开发是高度优先事项。为了开发这些策略，我们需要了解复制的基本生物学以及单个病毒基因在病毒生命周期中的功能。所有冠状病毒都含有结构蛋白，即刺突蛋白（S）、核衣壳蛋白（N）、膜蛋白（M）和小囊膜蛋白（E）。此外，每组冠状病毒都表达由小ORF编码的蛋白质，这是该组病毒所独有的。在某些情况下，这些对于复制是不必要的，但可能具有重要的辅助作用。因此，我们的目标是建立一个反向遗传学系统的SARS冠状病毒和选择病毒用于更详细的研究病毒的复制和发病机制。该项目的具体目标是：1）建立一个反向遗传学系统的SARS-CoV，使用靶向重组和2）确定小ORF编码的基因产物，独特的SARS-CoV，并探讨这些蛋白质在复制中的作用，并在长期的发病机制。 该反向遗传学系统将为SARS冠状病毒结构蛋白以及SARS冠状病毒小ORF中编码的小蛋白的结构/功能分析的长期研究提供基础。我们对冠状病毒的复制和发病机制有着长期的兴趣。 我们在这里提出的大部分工作都是基于我们在与小鼠冠状病毒，小鼠肝炎病毒（MHV）合作中积累的知识和专业知识。靶向重组系统极大地扩展了我们理解MHV复制和发病机制的能力，我们希望它也能对SARS-CoV起到同样的作用。

DESCRIPTION (provided by applicant): Within the last year a new coronavirus (SARS-CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS). Given the high mortality, the lack of effective treatment and the uncertainties of when the disease might return and at what frequency, a SARS vaccine and the development of antivirals are of high priority. In order to develop these strategies, we need to understand the basic biology of replication as well as the function of individual viral genes in the viral life cycle. All coronaviruses contain the structural proteins, spike (S), nucleocapsid (N), membrane (M) and small envelope (E). In addition, each group of coronaviruses expresses proteins encoded in small ORFs, unique to that group of viruses. These are, in some cases, nonessential for replication, but may have important accessory roles. Thus, our goals here include establishing a reverse genetics system for SARS-CoV and selecting viruses to be used for more detailed studies of viral replication and pathogenesis. The specific aims of this project are: 1) to establish a reverse genetics system for SARS-CoV, using targeted recombination and 2) to identify the gene products encoded in the small ORFs, unique to SARS-CoV, and to probe the role of these proteins in replication and, in the long term, in pathogenesis. The reverse genetics system will provide the basis for long-term studies on the structure/function analysis of viral structural proteins as well as the small proteins encoded in the small ORFs unique to SARS-CoV. We have a long-term interest in the replication and pathogenesis of coronaviruses. Much of the work we propose here is based on the knowledge and expertise we have accumulated in working with the murine coronavirus, mouse hepatitis virus (MHV). The targeted recombination system has greatly expanded our ability to understand replication and pathogenesis of MHV and we expect it will do the same for SARS-CoV.