Molecular Analysis of Alphavirus Membrane Fusion Protein

甲病毒膜融合蛋白的分子分析

• 批准号: 7919163
• 负责人: MARGARET KIELIAN
• 金额: $ 6.06万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-12 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7919163
• 关键词: Address; Alphavirus; Animals; Antiviral Therapy; Avidin; Back; Binding; Biochemical; Biotin; Carbohydrates; Categories; Cells; Cellular Membrane; Chimeric Proteins; Cholesterol; Collaborations; DNA Sequence Rearrangement; Dengue; Dengue Virus; Dependence; Dill; Eastern Equine Encephalomyelitis; Electron Microscopy; Encephalitis; Flavivirus; Grant; Histidine; Human; In Situ; Infection; Japanese Encephalitis; Knowledge; Length; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Molecular Analysis; Molecular Conformation; Mutagenesis; Negative Staining; Peptides; Polyarthritides; Positioning Attribute; Proteins; Reaction; Recombinants; Regulation; Resolution; Role; Semliki forest virus; Side; Site; Staging; Structure; Surface; System; Testing; Transmembrane Domain; Viral; Viral Fusion Proteins; Virus; Virus Diseases; West Nile virus; Work; Yellow Fever; base; chikungunya; design; dimer; driving force; expression cloning; inhibitor/antagonist; insight; pathogen; protein expression; research study; stem; three dimensional structure; tool; trimer core

DESCRIPTION (provided by applicant): Alphaviruses and flaviviruses cause severe human and animal illnesses such as encephalitis, polyarthritis, and dengue fever, with millions of cases in humans per year. These viruses include many potential bioterrorist agents that are category A, B, or C priority pathogens, such as the encephalitic alphaviruses and the flaviviruses West Nile, yellow fever, Japanese encephalitis and dengue virus (DV). Alphaviruses and flaviviruses infect cells through a low pH-triggered membrane fusion reaction mediated by their structurally similar fusion proteins. These "class II fusion proteins" rearrange to a target-membrane inserted homotrimer (HT) to drive the fusion reaction. In collaboration with Dr. Felix Rey, we have recently determined the structure of the HT of the fusion protein E1 from the alphavirus Semliki Forest virus (SFV). This structure reveals that the HT is a trimeric hairpin in which domain III (DIII) and the stem region fold back against the trimer core, positioning the fusion peptide loops and transmembrane (TM) domains at the same side of the molecule. We have developed recombinant DIll proteins that block refolding to the final hairpin and inhibit SFV and DV fusion and infection. Based on this progress, we will now address the molecular mechanism of class II membrane fusion using the highly developed SFV experimental system. We will characterize the critical features of trimerization using expressed E1 domains and structure-based mutagenesis studies. DIll proteins and a wide variety of available virus fusion intermediates will be used to define key conformational changes and correlate them with steps in fusion. All of the post-fusion structures of viral fusion proteins are missing the TM domain, and thus its interaction with the fusion loops is undefined. Through our ongoing collaboration we will determine the structure of the full-length E1 HT. We will use cryo- and negative stain electron microscopy to define the interaction of the fusion loop with the target membrane and the specific contacts between HTs. We will test the role of HT-interactions both at the fusion site and outside the fusion site to address the functions of HT cooperativity and "bystander" fusion proteins. Understanding the molecular mechanism of class II membrane fusion will provide insights into virus disease mechanisms, enable the design of specific antiviral therapies, and advance our knowledge of viral and cellular membrane fusion reactions.

描述（由申请人提供）：α病毒和黄病毒会导致严重的人类和动物疾病，例如脑炎，多关节炎和登革热，每年有数百万例患者。这些病毒包括许多潜在的生物恐怖剂，它们是A类，B或C优先病原体的许多潜在的生物恐怖剂，例如脑静脉α病毒和西尼罗河黄病毒，黄热病，日本脑炎和登革热病毒（DV）。 α病毒和黄病毒通过低pH触发的膜融合反应感染细胞，由它们的结构相似融合蛋白介导。这些“ II类融合蛋白”重新排列到目标膜插入的同型聚合物（HT）以驱动融合反应。与Felix Rey博士合作，我们最近确定了来自alphavirus semliki森林病毒（SFV）的融合蛋白E1 HT的结构。该结构表明，HT是一种三聚发夹，其中结构域III（DIII）和茎区域折叠回三聚体芯，将融合肽环和跨膜（TM）结构域定位在分子的同一侧。我们已经开发了重组莳萝蛋白，该蛋白会阻塞最终发夹，并抑制SFV，DV融合和感染。基于这一进展，我们现在将使用高度发达的SFV实验系统来解决II类膜融合的分子机制。我们将使用表达的E1结构域和基于结构的诱变研究来表征三聚化的关键特征。 DILL蛋白和各种可用的病毒融合中间体将用于定义关键构象变化，并将其与融合的步骤相关联。病毒融合蛋白的所有后融合结构都缺少TM结构域，因此其与融合环的相互作用均未定义。通过我们正在进行的协作，我们将确定全长E1 HT的结构。我们将使用冷冻染色电子显微镜来定义融合环与靶膜的相互作用以及HTS之间的特定接触。我们将测试HT相互作用在融合部位和融合位点外的作用，以解决HT协作性和“旁观者”融合蛋白的功能。了解II类膜融合的分子机制将提供对病毒疾病机制的见解，实现特定抗病毒药疗法的设计，并促进我们对病毒和细胞膜融合反应的了解。