Molecular Analysis of Alphavirus Membrane Fusion Protein

描述（由申请人提供）：甲病毒和黄病毒引起严重的人类和动物疾病，如脑炎、多关节炎和登革热，每年有数百万例人类病例。这些病毒包括许多潜在的生物恐怖主义制剂，属于A、B或C类优先病原体，如脑炎型甲病毒和黄病毒西尼罗河病毒、黄热病、日本脑炎和登革热病毒（DV）。甲病毒和黄病毒通过其结构相似的融合蛋白介导的低ph触发的膜融合反应感染细胞。这些“II类融合蛋白”重新排列成靶膜插入的同源三聚体（HT）来驱动融合反应。我们与Felix Rey博士合作，最近确定了来自塞姆利基森林病毒（SFV）的甲病毒融合蛋白E1的HT结构。这种结构揭示了HT是一个三聚体发夹，其中结构域III （DIII）和茎区向后折叠，使融合肽环和跨膜结构域位于分子的同一侧。我们已经开发了重组DIll蛋白，该蛋白阻断了向最终发夹的折叠，抑制了SFV和DV的融合和感染。基于这一进展，我们现在将利用高度发达的SFV实验系统来研究II类膜融合的分子机制。我们将利用表达E1域和基于结构的诱变研究来描述三聚化的关键特征。DIll蛋白和多种可用的病毒融合中间体将用于确定关键的构象变化并将其与融合步骤联系起来。所有病毒融合蛋白的融合后结构都缺少TM结构域，因此它与融合环的相互作用是不确定的。通过持续的合作，我们将确定全长E1 HT的结构。我们将使用低温和负染色电子显微镜来定义融合环与靶膜的相互作用以及热导膜之间的特定接触。我们将测试HT相互作用在融合位点和融合位点外的作用，以解决HT协同性和“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，使设计特异性抗病毒治疗成为可能，并提高我们对病毒和细胞膜融合反应的认识。

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.