GENETIC SYSTEM FOR CORONAVIRUSES

描述(改编自申请者摘要)：主要目标 这项建议是为了建立一个遗传系统，用于研究 冠状病毒的结构蛋白，一族单链阳性 其核衣壳组装成螺旋结构的感官RNA病毒。 该家族成员的rna基因组是最大的成熟rna。 尚未发现的分子，这使得冠状病毒的研究 已经被用于遗传研究的技术是不可接近的 操纵其他RNA病毒。已经开发了一个系统，用于 猪瘟病毒核衣壳蛋白基因的定点突变 冠状病毒原型，小鼠肝炎病毒(MHV)。此方法需要 它们的RNA-RNA重组率高的优势 通过以下途径将定点突变导入病毒基因组 与已被引入的合成RNA重组的方法 被感染的细胞。单点突变，以及广泛的替换， 已经以这种方式被改造成MHV。这一过程将是 扩展到研究病毒的其他结构蛋白：刺突 糖蛋白(S)、膜糖蛋白(M)和小膜 蛋白质(SM)，为阐明这些分子的结构特征提供工具 蛋白质和帮助确定它们在病毒中所扮演的角色 复制以及它们如何与病毒和宿主组件相互作用。在……里面 除了基于体内重组的MHV突变方法外， MHV基因组体外操作的多种方法将是 发展起来的。这些将使构建更大范围的突变体成为可能 类型，并将允许更高效和标准化的生产 重组病毒。拟议的研究将提供对 冠状病毒的生命周期和组装，抗病毒的潜在靶点 化疗，以及一种可能的手段来操纵这些感染性病原体 疫苗设计。

DESCRIPTION (Adapted from Applicant's Abstract): The major objective of this proposal is to establish a genetic system for the study of the structural proteins of coronaviruses, a family of single-stranded, positive sense RNA viruses whose nucleocapsids are assembled into helical structures. The RNA genomes of members of this family are the largest mature RNA molecules yet discovered, which has made the study of coronaviruses unapproachable by the techniques that have been used for the genetic manipulation of other RNA viruses. A system has been developed for the site-specific mutagenesis of the nucleocapsid (N) protein gene of the prototype coronavirus, mouse hepatitis virus (MHV). This method takes advantage of the high rate of RNA-RNA recombination characteristic of these viruses by transducing a site-specific mutation into the viral genome by means of recombination with a synthetic RNA that has been introduced into infected cells. Single point mutations, as well as extensive substitutions, have been engineered into MHV in this fashion. This procedure will be extended to study the other structural proteins of the virus: the spike glycoprotein (S), the membrane glycoprotein (M), and the small membrane protein (sM), to provide tools for elucidating structural features of these proteins and for helping to determine the roles they play in viral replication and how they interact with both viral and host components. In addition to in vivo recombination based approaches to MHV mutagenesis, multiple methods for the in vitro manipulation of the MHV genome will be developed. These will enable the construction of a wider range of mutant types and will allow more efficient and standardized production of recombinant viruses. The proposed studies will provide insights into the coronavirus life cycle and assembly, potential targets for antiviral chemotherapy, and a possible means to manipulate these infectious agents for vaccine design.