Inhibition of the Membrane Fusion Proteins of Flaviviruses and Alphaviruses

描述(申请人提供)：黄病毒和甲型病毒通过蚊子和扁虱媒介传播，并导致严重的人类和动物疾病，如脑炎和出血热。这些病毒包括许多潜在的生物恐怖分子，属于A-C类病原体，如黄病毒登革热、西尼罗河病毒、日本脑炎和黄热病病毒，以及委内瑞拉、东部和西部马脑炎病毒的甲型病毒。登革热病毒(DV)目前特别令人担忧，因为它已经戏剧性地重新出现，在包括美国在内的100多个国家成为地方性流行病，现在是一个全球卫生问题。据估计，每年有5 000万至1亿登革热病例和500 000例更致命的并发症登革热出血热，对人类健康和发展中国家的经济都有重大影响。迫切需要针对黄病毒和甲型病毒的抗病毒策略。黄病毒和甲病毒膜融合蛋白是II类病毒融合蛋白的成员。它们在结构上非常相似，并重新折叠为同源三聚体形式，以介导病毒融合和感染。最近，我们与Félix Rey博士合作，确定了甲型病毒塞姆利基森林病毒(SFV)融合蛋白的同源三聚体构象的结构。SFV的同源三聚体结构与DV的结构惊人地相似。以该结构为指导，我们开发出了作为SFV和DV融合和感染的特异性显性-负性抑制因子的蛋白质片段。基于这些信息，我们现在计划开发一种通用的II类融合反应抑制剂的筛查方法。在目标1中，我们将建立和优化体外方法，以跟踪II类融合蛋白复性反应中蛋白质-蛋白质的相互作用。在目标2中，我们将使这种分析方法适应高通量格式，并使用它来筛选我们研究所以及NIH和东北生物防御联盟提供的多肽和小分子文库。最终，这些抑制剂将成为抗病毒治疗的先导化合物，以及理解II类病毒融合反应的重要研究工具。黄病毒和甲病毒包括许多重要的人类病原体和潜在的生物恐怖威胁，如黄病毒登革热、西尼罗河、日本脑炎和黄热病病毒，以及甲病毒委内瑞拉、东部和西部马脑炎病毒。登革热病毒目前尤其令人担忧，因为它已经戏剧性地卷土重来，在包括美国在内的100多个国家流行，现在是一个全球健康问题。这项应用侧重于开发针对黄病毒和甲型病毒的新的抗病毒策略，其基础是阻止病毒最初进入细胞所涉及的蛋白质的活性。

DESCRIPTION (provided by applicant): Flaviviruses and alphaviruses are spread by mosquito and tick vectors and cause severe human and animal illnesses such as encephalitis and hemorrhagic fever. These viruses include many potential bioterrorist agents that are category A-C pathogens, such as the flaviviruses dengue, West Nile, Japanese encephalitis and yellow fever viruses, and the alphaviruses Venezuelan, eastern, and western equine encephalitis viruses. Dengue virus (DV) is currently of particular concern as it has dramatically reemerged to become endemic in more than 100 countries including the US, and is now a global health problem. There are an estimated 50-100 million cases of dengue fever and 500,000 cases of the more lethal complication dengue hemorrhagic fever per year, with significant impact on both human health and the economies of developing countries. Antiviral strategies for the flaviviruses and alphaviruses are urgently needed. The flavivirus and alphavirus membrane fusion proteins are members of the class II virus fusion proteins. They are structurally very similar and refold to a homotrimer form to mediate virus fusion and infection. In collaboration with Dr. F¿lix Rey, we have recently determined the structure of the homotrimer conformation of the fusion protein from the alphavirus Semliki Forest virus (SFV). The SFV homotrimer structure is strikingly similar to that of DV. Using the structure as a guide, we have developed protein fragments that act as specific dominant-negative inhibitors of SFV and DV fusion and infection. Based on this information, we now plan to develop a general screen for inhibitors of class II fusion reactions. In aim 1 we will establish and optimize in vitro methods to follow the protein-protein interactions during the class II fusion protein refolding reaction. In aim 2, we will adapt this assay to a high throughput format, and use it to screen peptide and small molecule libraries available at our institution and through the NIH and Northeast Biodefense Consortium. Ultimately, such inhibitors will be lead compounds for antiviral therapy, and important research tools to understand the class II virus fusion reaction. Flaviviruses and alphaviruses include many important human pathogens and potential bioterrorist threats such as the flaviviruses dengue, West Nile, Japanese encephalitis and yellow fever viruses, and the alphaviruses Venezuelan, eastern, and western equine encephalitis viruses. Dengue virus is currently of particular concern as it has dramatically reemerged to become endemic in more than 100 countries including the US, and is now a global health problem. This application focuses on developing new antiviral strategies for the flaviviruses and alphaviruses, based on blocking the activity of the proteins involved in the initial entry of the virus into the cell.