CORONAVIRUS RNA SYNTHESIS

这项建议的主要目的是利用遗传学和生物化学方法来研究冠状病毒中RNA复制和转录的独特机制。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分子之一，这使得它们的遗传操作是其他RNA病毒所使用的技术所无法比拟的。已开发出一种替代系统，靶向RNA重组，用于对原型冠状病毒小鼠肝炎病毒(MHV)的所有结构基因进行定点突变。这种方法利用了MHV中RNA-RNA的高重组率，通过与被感染细胞中的合成RNA重组，将定点突变转移到病毒基因组中。单点突变和广泛的替换已经以这种方式被改造成MHV。这个程序现在将被用来研究基因组元件在病毒RNA合成中所起的作用。首先，将探索病毒基因组3‘非翻译区功能元件的结构和作用方式，以了解它们如何参与复制。其次，将研究序列的作用和基因间元件的背景，以了解病毒信使RNA是如何产生的。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 use genetic and biochemical methods to study the unique mechanism of RNA replication and transcription in coronaviruses. Coronaviruses are a family of single-stranded, positive-sense RNA viruses whose helical nucleocapsids are packaged within host-derived membrane envelopes containing a small complement of viral membrane glycoproteins. The RNA genomes of coronaviruses are among the largest mature RNA molecules yet discovered, making their genetic manipulation unapproachable by the techniques that have been used with other RNA viruses. An alternative system, targeted RNA recombination, has been developed for the site-specific mutagenesis of all the structural genes of the prototype coronavirus mouse hepatitis virus (MHV). This method takes advantage of the high rate of RNA-RNA recombination in MHV by transducing site-specific mutations into the viral genome by recombination with a synthetic RNA introduced into infected cells. Single point mutations and extensive substitutions have been engineered into MHV in this fashion. This procedure will now be used to study the roles played by genomic elements in viral RNA synthesis. First, the structure and mode of action of functional elements in the 3' untranslated region of the viral genome will be explored to learn how they participate in replication. Second, the roles of the sequence and the context of intergenic elements will be examined to gain an understanding of how viral messenger RNAs are produced. 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 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.