A GENETIC SYSTEM FOR CORONAVIRUSES

这项建议的主要目的是进一步开发和应用研究冠状病毒的遗传系统。冠状病毒是一类单链、阳性的RNA病毒，其螺旋核衣壳被包装在宿主衍生的含有少量病毒膜糖蛋白的膜囊膜中。冠状病毒的RNA基因组是迄今发现的最大的成熟RNA分子，这使得它们的遗传操作是其他RNA病毒所使用的技术无法达到的。我们已经建立了一种对原型冠状病毒小鼠肝炎病毒(HMV)进行定点突变的系统，该系统利用了MHV中RNA-RNA重组率高的优点，通过与被感染细胞中的合成RNA重组将定点突变导入病毒基因组中。单点突变和广泛的替换已经被以这种方式工程到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所提供的优势以及获得具有感染力的MHV基因组全长cDNA的努力将进一步加强这一程序。该遗传系统将被用来回答有关MHV病毒粒子成分的结构和功能的基本问题：尖峰糖蛋白、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扮演的角色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，了解它们的分子生物学对于控制和预防它们至关重要。建议的研究将提供对冠状病毒生命周期和组装的基本见解，抗病毒化疗的潜在靶点，以及为疫苗设计操纵这些感染性病原体的可能方法。

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.