Genetic Analysis of Molecular Interactions in Coronavirus Replication

描述（由申请方提供）：冠状病毒是一个包膜RNA病毒家族，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道综合征（SARS）的病原体，SARS有可能从动物宿主重新出现在人群中。该提案的总体目标是使用原型冠状病毒小鼠肝炎病毒（MHV）描述对冠状病毒复制的多个阶段至关重要的机制。冠状病毒在所有RNA病毒中具有最大的基因组，因此其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最早阶段，以及在感染的晚期，当子代包膜蛋白和核衣壳联合收割机结合以出芽时，我们对这些基本事件的了解仍存在许多空白。为了操纵冠状病毒的基因组，我们通过靶向RNA重组开发了最早的反向遗传系统。这种强大而通用的技术已成为分析MHV结构蛋白的不可或缺的工具。最近，全长冠状病毒cDNA系统也已可用，允许获得病毒复制酶。这些强大的反向遗传学方法，加上互补的生物化学和分子生物学方法，将用于实现三个具体目标。(1)我们将研究一个新发现的核衣壳（N）蛋白和病毒复制-转录复合体（nsp 3）的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nsp 3分子的特定模块。(2)我们将阐明N蛋白在冠状病毒感染起始中的关键作用，我们假设，这是将病毒基因组传递到新生的复制-转录复合体。此外，我们将了解如何通过N蛋白的磷酸化来调节这种作用。(3)我们将进一步剖析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的网络，是必不可少的病毒体组装。这些努力将建立在我们发现的不寻常的M蛋白变异，在E缺失突变体的演变，并将使用种间M蛋白嵌合体探测M-M和M-N相互作用。此外，我们将探讨基因组包装信号在选择性地将核衣壳并入病毒体中的作用。

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.