Initiation of Nucleocapsid Assembly in Ebola virus

描述(申请人提供)：埃博拉出血热是最致命的传染病之一。其病原体埃博拉病毒被归类为一种新出现的传染病，一种可能用于生物恐怖主义的A类生物制剂，以及一种精选制剂。尽管有这些相当戏剧性的事实，但目前还没有治疗这种疾病的方法。这种需求和供应之间的差异在很大程度上是因为我们对埃博拉病毒的基本生物学缺乏了解。 埃博拉病毒属于丝状病毒科。它由一条约19千碱基的负义RNA链组成，紧密包裹在病毒蛋白中。这种核糖核蛋白复合体的形成被称为核衣壳，假设始于病毒核衣壳蛋白和复制的病毒RNA的5‘末端之间的相互作用。指导这种相互作用的具体机制尚不清楚。 这一应用程序开始解决了解埃博拉病毒包囊的整体机制这一长期目标。具体地说，将确定病毒RNA和核衣壳蛋白中促进核衣壳形成的残基。将建立NP-RNA结合试验来测量这种相互作用的解离常数。RNA和蛋白质的初步截断将建立各自结合所必需的区域。交联性实验也将被用于这一分析。然后，定向突变将分离RNA和蛋白质中错综复杂地参与核衣壳形成的残基。这些体外研究的结果将在重组的病毒复制系统中进行测试。微型复制系统和完整的病毒反向遗传学系统都可用于这些实验，在该系统中，整个病毒由质粒重组。这项研究的结果将是重要的，因为它将填补我们对埃博拉病毒复制知识的空白，从而导致未来旨在抑制包膜的抗病毒疗法的发展，包膜是埃博拉病毒生命周期中一个重要但鲜为人知的步骤。

DESCRIPTION (provided by applicant): Ebola hemorrhagic fever is among the deadliest of communicable diseases. Its causative agent, Ebola virus, is categorized as both an emerging infectious disease, a Class A biological agent with potential for use in bioterrorism, and a Select Agent. Despite these rather dramatic facts, there are currently no available treatments for people suffering with this disease. This discrepancy between need and supply is in large part due to our lack of understanding of the basic biology of Ebola virus. Ebola virus is classified in the family Filoviridae. It consists of a single, approximately 19-kilobase strand of negative-sense RNA that is tightly wrapped in viral proteins. Formation of this ribonucleoprotein complex, referred to as the nucleocapsid, is hypothesized to commence with interactions between the viral nucleocapsid protein and the 5' termini of the replicating viral RNA. The specific mechanisms that guide this interaction are unknown. This application begins to address the long-term goal of understanding the overall mechanism of encapsidation in Ebola virus. Specifically, residues within the viral RNA and the nucleocapsid protein that facilitate nucleocapsid formation will be identified. An NP-RNA binding assay will be established to measure the dissociation constant for this interaction. Preliminary truncations of the RNA and the protein will establish regions of each that are necessary for binding. Cross-linking experiments will also be utilized for this analysis. Directed mutagenesis will then isolate residues within the RNA and the protein that are intricately involved in nucleocapsid formation. The results of these in vitro studies will be tested in reconstituted viral replication systems. Both mini-replication systems and a full viral reverse genetics system, in which the entire virus is reconstituted from plasmids, are available for these experiments. The outcome from this research will be significant as it will fill a gap in our knowledge of the replication of Ebola virus and thus lead to the future development of antiviral therapies aimed at inhibition of encapsidation, an essential, but poorly understood step in the lifecycle of Ebola virus.