Molecular Basis for Ebola Virus Pathogenicity

描述（由申请人提供）：本研究的长期目标是在分子水平上了解埃博拉病毒感染的发病机制。作为一种潜在的生物恐怖主义制剂，埃博拉病毒会导致人类严重的出血性疾病，是迄今为止遇到的最致命的人类病原体之一，在NIAID生物防御和新兴传染病研究机会计划中被列为“A类优先病原体”。然而，既没有治疗方法，也没有保护性疫苗来控制这种往往致命的感染。完全从克隆cDNA中产生埃博拉病毒的反向遗传学系统和生物安全4级设施的结合使申请人能够实现以下具体目标：1)了解埃博拉病毒转录的调控，其中将使用双链微型复制子和反向遗传学策略来评估埃博拉病毒基因组独特特征的复制意义，例如重叠的转录停止/启动信号和异常长的基因间区域；2)明确传染性埃博拉病毒颗粒形成的驱动机制，研究VP40基质蛋白和GP糖蛋白在颗粒形成和出芽中的作用；3)明确VP24蛋白在埃博拉病毒复制中的作用，确定VP24在细胞内的定位，与细胞膜和/或其他病毒蛋白的结合，以及在病毒粒子出芽和膜融合中的功能；4)确定导致埃博拉病毒感染极端毒力的因素和机制，其中将使用小鼠埃博拉病毒感染模型筛选可能增强或减弱病毒致病性的突变。此外，使用野生型埃博拉病毒，也将评估furin介导的GP切割或GP中的粘蛋白样结构域以及分泌的糖蛋白sGP对埃博拉毒力的可能贡献。总的来说，这些研究将有助于破译这种病原体的极端毒力的分子基础。最终，获得的信息可用于开发抗病毒化合物和有效疫苗。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the pathogenesis of Ebola virus infection at the molecular level. A potential bioterrorism agent, Ebola virus causes severe hemorrhagic disease in humans and is one of the most deadly human pathogens encountered to date, earning classification as a 'Category A Priority Pathogen' in the NIAID Biodefense and Emerging Infectious Disease Research Opportunities Program. Yet, neither therapeutic treatments nor protective vaccines are available to control this often lethal infection. The combination of a reverse genetics system for the generation of Ebola virus entirely from cloned cDNA and access to a biosafety level 4 facility has positioned the applicant to undertake the following specific aims: 1) To understand the regulation of Ebola viral transcription, in which bicistronic minireplicon and reverse genetics strategies will be used to assess the replicative significance of the unique features characterizing the Ebola virus genome, such as overlapping transcriptional stop/start signals and an unusually long intergenic region; 2) To clarify the mechanisms that drive the formation of infectious Ebola virus particles, in which the roles of the VP40 matrix protein and GP glycoprotein in particle formation and budding will be investigated; 3) To define the role of VP24 protein in Ebola viral replication, in which the intracellular localization of VP24, its association with cellular membranes and/or other viral proteins, and its function in virion budding and membrane fusion will be determined; and 4) To identify the factors and mechanisms responsible for the extreme virulence of Ebola virus infection, in which a murine model of Ebola virus infection will be used to screen mutations that may enhance or diminish viral pathogenicity. Moreover, using a wild-type Ebola virus, furin-mediated GP cleavage, or the mucin-like domain in GP, and the secreted glycoprotein, sGP, will also be evaluated for a possible contribution to Ebola virulence. Collectively, these studies will contribute to the deciphering of the molecular basis for the extreme virulence of this pathogen. Ultimately, the information gained could be exploited in the development of antiviral compounds and effective vaccines.