Mechanism and inhibition of dengue and chikungunya virus fusion protiens

登革热和基孔肯雅病毒融合蛋白的作用机制及抑制作用

• 批准号: 7670803
• 负责人: MARGARET KIELIAN
• 金额: $ 68.13万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7670803
• 关键词: Abbreviations; Alphavirus; Animals; Antiviral Agents; Antiviral Therapy; Binding; Biological Assay; Case Study; Categories; Chikungunya virus; Chimeric Proteins; Collaborations; Country; Culicidae; Dengue; Dengue Virus; Dill; Disease Outbreaks; Dominant-Negative Mutation; Encephalitis; Flavivirus; Flavivirus Infections; Fluorescence; Goals; Grant; Health; Human; India; Infection; Lead; Mediating; Membrane Fusion; Molecular Conformation; Proteins; Reaction; Recombinant Proteins; Recombinants; Research; Semliki forest virus; Site; Specificity; Structure; System; Testing; Ticks; United States National Institutes of Health; Viral; Viral Hemorrhagic Fevers; Virus; Virus Diseases; Virus Inhibitors; base; biodefense; high throughput screening; in vitro Assay; inhibitor/antagonist; member; novel; pathogen; small molecule; small molecule libraries; tool; trimer core; vector

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.

黄病毒和甲病毒通过蚊子和蜱媒传播，并引起严重的人类和 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，如黄病毒登革热病毒（DV）和甲病毒属 基孔肯雅病毒（CV）。家庭暴力特别令人关切，因为它在非洲急剧重新出现，成为地方病。 包括美国在内的100多个国家，现在是一个全球性的健康问题。甲病毒也可以是 重要的新兴病原体，例如最近爆发的CV感染， 迄今为止在印度报告的病例。迫切需要针对黄病毒和甲病毒的抗病毒策略。 黄病毒和甲病毒膜融合蛋白是II类病毒膜的成员 融合蛋白它们在结构上非常相似，并重折叠成同源三聚体形式以介导病毒融合， 感染与Felix Rey博士合作，我们确定了 来自甲病毒属塞姆利基森林病毒（SFV）的融合蛋白。SFV同源三聚体结构是惊人的 类似于DV。利用这些结构作为指导，我们最近开发了重组形式的 来自SFV和DV融合蛋白的结构域III（DUN）。这些重组Dill蛋白作为特异性的 病毒融合和感染的显性负性抑制剂，是我们提出的筛选小 DV和CV的分子抑制剂。 目的1：利用我们的重组蛋白系统来阐明DV和CV融合的机制 蛋白质三聚化和三聚体形成的步骤。在aim 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测定。在目标3中，我们将使该测定适应于高浓度。 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测定法 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命中 和感染最终，这种抑制剂将成为抗病毒治疗的先导化合物， 了解II类病毒融合反应的工具。