Mechanism and inhibition of dengue and chikungunya virus fusion protiens

黄病毒和甲型病毒通过蚊子和扁虱媒介传播，导致严重的人类和 动物疾病，如脑炎和出血热。这些病毒包括潜在的生物恐怖分子 A-C类病原体，如黄病毒登革病毒(DV)和甲型病毒 基孔肯雅病毒(CV)。DV尤其令人担忧，因为它已戏剧性地重新出现，成为#年的地方性流行病。 包括美国在内的100多个国家，现在是一个全球健康问题。甲型病毒也可以是 重要的新兴病原体，例如最近爆发的数百万冠状病毒感染 印度迄今报告了病例。迫切需要针对黄病毒和甲型病毒的抗病毒策略。 黄病毒和甲型病毒膜融合蛋白是第二类病毒膜的成员。 融合蛋白。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。与Felix Rey博士合作，我们确定了同源三聚体构象的结构 甲型病毒塞姆利基森林病毒(SFV)的融合蛋白。SFV均三聚体结构惊人地 与DV相似。以这些结构为指导，我们最近开发出了重组形式的 SFV和DV融合蛋白的结构域III(Dun)。这些重组Dill蛋白作为特定的 显性-阴性的病毒融合和感染抑制物，是我们建议的小样本筛查的基础 DV和CV的分子抑制剂。 在目标1中，我们将使用我们的重组蛋白系统来确定DV和CV融合的机制 蛋白质三聚及形成三聚体的步骤。在目标2中，我们将在体外开发基于荧光的 DIM与DV和CV的三聚体核心结合的分析。在目标3中，我们将把这种分析调整到一个高水平 吞吐量格式。然后，我们将在小分子文库的高通量筛选中使用该分析 可通过NIH和东北生物防御联盟获得，并通过病毒融合测试验证HITS 和感染。最终，这些抑制剂将成为抗病毒治疗的先导化合物，并进行重要的研究 了解II类病毒融合反应的工具。

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 potential bioterrorist agents that are category A-C pathogens, such as the flavivirus dengue virus (DV) and the alphavirus Chikungunya virus (CV). DV is 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. Alphaviruses can also be important emerging pathogens, as exemplified by the recent outbreak of CV infection with several million cases reported to date in India. Antiviral strategies for the flaviviruses and alphaviruses are urgently needed. The flavivirus and alphavirus membrane fusion proteins are members of the class II virus membrane fusion proteins. They are structurally very similar and refold to a homotrimer form to mediate virus fusion and infection. In collaboration with Dr. Felix Rey, we 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 structures as a guide, we have recently developed recombinant forms of domain III (DUN) from the SFV and DV fusion proteins. These recombinant Dill proteins act as specific dominant-negative inhibitors of virus fusion and infection, and are the basis for our proposed screen for small molecule inhibitors of DV and CV. In aim 1 we will use our recombinant protein systems to define the mechanism of DV and CV fusion protein trimerization and the steps in trimer formation. In aim 2, we will develop fluorescence-based in vitro assays for the binding of DIM to the trimer core of DV and CV. In aim 3 we will adapt this assay to a high throughput format. We will then use the assay in high throughput screens of small molecule libraries available through the NIH and the Northeast Biodefense Consortium, and validate hits by tests of virus fusion and infection. Ultimately, such inhibitors will be lead compounds for antiviral therapy, and important research tools to understand the class II virus fusion reaction.