GENETIC ANALYSIS OF CORONAVIRUS ASSEMBLY INTERACTIONS

描述（由申请人提供）：冠状病毒是一类包膜RNA病毒，可在哺乳动物和鸟类宿主中引起呼吸道、肠道和神经系统疾病。在人类中，有三种冠状病毒导致上呼吸道感染；第四种人类冠状病毒是最近发现的SARS病原体。冠状病毒的基因组是所有RNA病毒中最大的，这使得其基因操作成为一个艰巨的问题。我们的实验室开发了最早的冠状病毒反向遗传系统，称为靶向RNA重组，与原型冠状病毒小鼠肝炎病毒（MHV）。该方法利用MHV的高RNA重组率，通过将合成RNA引入感染细胞重组，将位点特异性突变转导到病毒基因组中。再加上强大的宿主范围选择系统，靶向RNA重组已成为一种强大而通用的技术，已被用于回答有关病毒蛋白结构和功能、宿主物种特异性、病毒粒子组装以及冠状病毒RNA合成复杂机制的基本问题。本提案的主要目的是进一步扩展冠状病毒组装机制的遗传学研究。靶向RNA重组和互补生化分析将用于回答MHV结构蛋白在病毒复制中的功能作用的基本问题：小包膜蛋白如何与膜蛋白合作驱动病毒包膜的形成和出芽；刺突糖蛋白内结构域如何指定其在病毒粒子组装中的内含物；以及核衣壳蛋白如何与基因组RNA结合并被选择并入病毒粒子。了解冠状病毒的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、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。

DESCRIPTION (provided by applicant): Coronaviruses are a family of enveloped RNA viruses that cause respiratory, enteric, and neurologic diseases in mammalian and avian hosts. In humans, three coronaviruses are responsible for upper respiratory tract infections; a fourth human coronavirus is the recently discovered causative agent of SARS. The genomes of coronaviruses are the largest among all the RNA viruses, which has made their genetic manipulation a formidable problem. Our laboratory developed the earliest reverse genetic system for coronaviruses, called targeted RNA recombination, with the prototype coronavirus mouse hepatitis virus (MHV). This method exploits the high rate of RNA recombination in MHV to transduce site-specific mutations into the viral genome by recombination with synthetic RNA introduced into infected cells. Coupled with a powerful host-range-based selection system, targeted RNA recombination has become a robust and versatile technique that has been used to answer fundamental questions about viral protein structure and function, host species specificity, virion assembly, and the complex mechanism of coronavirus RNA synthesis. The major object of this proposal is to further extend our genetic studies of coronavirus assembly mechanisms. Targeted RNA recombination and complementary biochemical analyses will be used to answer basic questions about the functional roles of MHV structural proteins in viral replication: how the small envelope protein cooperates with the membrane protein to drive formation and budding of the virus envelope; how the spike glycoprotein endodomain specifies its inclusion in virion assembly; and how the nucleocapsid protein binds to genomic RNA and is selected for incorporation into virions. An understanding of the molecular biology of coronaviruses is critical for their control and prophylaxis. The proposed studies will provide fundamental insights into the coronavirus life cycle, potential targets for antiviral chemotherapy, and a possible means to manipulate these infectious agents for vaccine design.