A GENETIC SYSTEM FOR CORONAVIRUSES

本提案的主要目的是进一步开发和应用冠状病毒研究的遗传系统。冠状病毒是一类单链阳性RNA病毒，整个螺旋状核衣壳被包装在宿主衍生的膜包膜内，内含少量病毒膜糖蛋白。冠状病毒的RNA基因组是迄今为止发现的最大的成熟RNA分子，这使得它们的基因操作是其他RNA病毒所使用的技术无法接近的。我们建立了一种原型冠状病毒小鼠肝炎病毒（HMV）的位点特异性突变系统，该系统利用MHV中RNA-RNA高重组率的优势，通过将合成RNA引入感染细胞中重组，将位点特异性突变转导到病毒基因组中。单点突变和广泛的替换已经以这种方式被设计到MHV的所有结构蛋白基因中。强大的基于宿主范围的选择系统提供的优势以及获得MHV基因组的感染性全长cDNA的努力将进一步增强这一过程。遗传系统将用于回答MHV病毒粒子组成部分的结构和功能的基本问题：刺突糖蛋白、基质糖蛋白、小包膜蛋白和核衣壳蛋白。这将为这些蛋白质在病毒复制中的作用以及它们如何与病毒和宿主成分相互作用提供有价值的见解。冠状病毒是重要的呼吸道、神经系统和肠道病原体，了解其分子生物学对其控制和预防至关重要。拟议的研究将为冠状病毒的生命周期和组装、抗病毒化疗的潜在靶点以及操纵这些感染因子进行疫苗设计的可能手段提供基本见解。

The major object of this proposal is to further develop and apply a genetic system for the study of coronaviruses. Coronoaviruses are a family of single-stranded, positive-sense RNA viruses whoe helical nucleocapsids are packaged within host-derived membrane envelopes containing a small complement of viral membrane glycoproteins. The RNA genomes of coronaviruses are the largest mature RNA molecules yet discovered, making their genetic manipulation unapproachable by the techniques that have been used with other RNA viruses. We have established a system for the site-specific mutagenesis of the prototype coronavirus mouse hepatitis virus (HMV) which takes advantage of the high rate of RNA-RNA recombination in MHV by transducing a site-specific mutation into the viral genome by recombination with a synthetic RNA introduced into infected cells. Single point mutations and extensive substitutions have been engineered into all of the structural protein genes of MHV in this fashion. This procedure will be enhanced further by the advantages offered by a powerful host-range-based selection system and by efforts to obtain an infectious full-length cDNA of the MHV genome. The genetic system will be used to answer basic questions about the structure and function of the components of MHV virions: the spike glycoprotein, the matrix glycoprotein, the small envelope protein, and the nucleocapsid protein. This will provide valuable insights into the roles these proteins play in viral replication and how they interact with both viral and host components. Coronaviruses are important respiratory, neurologic and enteric pathogens, and an understanding of their molecular biology is critical for their control and prophylaxis. The studies proposed will provide fundamental 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.