Genetic Analysis of Molecular Interactions in Coronavirus Replication

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，四种冠状病毒是常见的上呼吸道感染的原因；第五种人类冠状病毒是严重急性呼吸综合征(SARS)的病原体，它有可能从动物宿主重新出现在人群中。这项建议的总体目标是利用冠状病毒小鼠肝炎病毒(MHV)的原型来描述对冠状病毒复制的多个阶段至关重要的机制。冠状病毒拥有所有RNA病毒中最大的基因组，因此它们的基本分子生物学是复杂的。因此，在病毒核衣壳进入宿主细胞后的感染早期阶段，以及在感染的后期，当后代包膜蛋白和核衣壳蛋白结合发芽时，我们对基本事件的了解仍然存在许多空白。为了操纵冠状病毒的基因组，我们开发了最早的反向遗传系统，通过靶向RNA重组。这项强大而通用的技术已经成为分析MHV结构蛋白不可或缺的工具。最近，全长冠状病毒cdna系统也已可用，允许访问病毒复制酶。这些强大的反向遗传方法，加上互补的生化和分子生物学方法，将被用来实现三个具体目标。(1)我们将研究新发现的核衣壳(N)蛋白与病毒复制-转录复合体(NSP3)的一个组成部分之间的关键相互作用。这将需要识别对病毒复制至关重要的巨大nsp3分子的特定模块。(2)我们将阐明N蛋白在冠状病毒感染启动中的关键作用，我们假设，N蛋白是将病毒基因组传递到新生的复制-转录复合体。此外，我们还将学习N蛋白的磷酸化是如何调节这种作用的。(3)我们将进一步剖析病毒粒子组装所必需的N蛋白、膜(M)蛋白和小包膜(E)蛋白之间的结构蛋白相互作用网络。这些努力将建立在我们发现在E缺失突变体中进化的不寻常的M蛋白变体的基础上，并将使用物种间M蛋白嵌合体来探索M-M和M-N相互作用。此外，我们还将探索基因组包装信号在选择性地将核衣壳掺入病毒粒子中的作用。

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, four coronaviruses are responsible for common upper respiratory tract infections; a fifth human coronavirus is the causative agent of severe acute respiratory syndrome (SARS), which has the potential to re-emerge into the population from animal reservoirs. The overall goal of this proposal is to delineate mechanisms that are critical to multiple phases of coronavirus replication, using the prototype coronavirus mouse hepatitis virus (MHV). Coronaviruses have the largest genomes of all RNA viruses and their basic molecular biology is consequently intricate. There thus remain numerous gaps in our knowledge of the essential events at the earliest stages of infection, following entry of the viral nucleocapsid into the host cell, and at late stages of infection, when progeny envelope proteins and nucleocapids combine for budding. To manipulate the genomes of coronaviruses, we developed the earliest reverse genetic system, via targeted RNA recombination. This robust and versatile technique has been an indispensable tool for analyses of MHV structural proteins. More recently, full-length coronavirus cDNA systems have also become available, allowing access to the viral replicase. These powerful reverse genetic methodologies, together with complementary biochemical and molecular biological approaches, will be employed to accomplish three specific aims. (1) We will investigate a newly discovered critical interaction between the nucleocapsid (N) protein and a component of the viral replication-transcription complex (nsp3). This will entail identification of the particular modules of the huge nsp3 molecule that are essential for viral replication. (2) We will elucidate the crucial role of N protein in the initiation of coronavirus infection, which, we hypothesize, is to deliver the viral genome to the nascent replication-transcription complex. Moreover, we will learn how this role is modulated by phosphorylation of N protein. (3) We will further dissect the network of structural protein interactions among N protein, the membrane (M) protein, and the small envelope (E) protein that are essential to virion assembly. These efforts will build upon our discovery of unusual M protein variants that evolve in E deletion mutants, and will use interspecies M protein chimeras to probe M-M and M-N interactions. Additionally, we will explore the role of the genomic packaging signal in the selective incorporation of the nucleocapsid into virions.