GENETIC SYSTEM FOR CORONAVIRUSES

描述（改编自申请人的摘要）： 这项建议是建立一个遗传系统的研究， 冠状病毒的结构蛋白，一个单链，阳性 有义RNA病毒，其核衣壳装配成螺旋结构。 该家族成员的RNA基因组是最大的成熟RNA 这使得对冠状病毒的研究 无法通过用于遗传学的技术 操纵其他RNA病毒。 已经开发了一个系统， 核衣壳（N）蛋白基因的位点特异性诱变 原型冠状病毒，小鼠肝炎病毒（MHV）。 该方法以 这些基因的高RNA-RNA重组率特征的优点是 通过将位点特异性突变转导到病毒基因组中， 与已被引入的合成RNA重组的方法 被感染的细胞 单点突变，以及广泛的取代， 都是以这种方式被设计到MHV中的。 本程序将 扩展到研究病毒的其他结构蛋白： 糖蛋白（S）、膜糖蛋白（M）和小膜糖蛋白（M）。 蛋白质（sM），以提供工具，阐明这些结构特征， 蛋白质，并帮助确定它们在病毒中发挥的作用， 复制以及它们如何与病毒和宿主成分相互作用。 在 除了基于体内重组的MHV诱变方法之外， 多种体外操作MHV基因组的方法将被 开发 这将使构建更广泛的突变体成为可能。 类型，并将允许更高效和标准化的生产 重组病毒 拟议的研究将提供深入了解 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因子， 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.