GENETIC ANALYSIS OF CORONAVIRUS ASSEMBLY INTERACTIONS

描述(申请人提供)：冠状病毒是一类囊膜RNA病毒，可引起哺乳动物和禽类宿主的呼吸道、肠道和神经系统疾病。在人类中，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的病原体。冠状病毒的基因组是所有RNA病毒中最大的，这使得它们的遗传操作成为一个可怕的问题。我们实验室以冠状病毒小鼠肝炎病毒(MHV)为原型，开发了最早的冠状病毒反向遗传系统，称为靶向RNA重组。这种方法利用MHV的高RNA重组率，通过与被感染细胞中的合成RNA重组，将定点突变转导到病毒基因组中。与强大的基于宿主范围的选择系统相结合，靶向RNA重组已成为一种强大而通用的技术，已被用于回答病毒蛋白质结构和功能、宿主物种特异性、病毒粒子组装以及冠状病毒RNA合成的复杂机制等基本问题。这项建议的主要目的是进一步扩大我们对冠状病毒组装机制的遗传学研究。有针对性的RNA重组和互补的生化分析将被用来回答关于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来驱动病毒包膜的形成和萌发；刺状糖蛋白内域如何指定其包含在病毒粒子组装中；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中。了解冠状病毒的分子生物学对于其控制和预防至关重要。拟议的研究将提供对冠状病毒生命周期的基本见解，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。

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.