Flavivirus RNA replication proteins: novel targets for drug discovery

登革热病毒和西尼罗河病毒是由蚊子传播的黄病毒，具有相当大的潜力作为 生物恐怖主义。这些正链rna病毒也是与自然疾病有关的新病原体。 暴发，使其控制成为全世界一个重要的公共卫生目标(特别是登革热病毒)。在……里面 除了针对病毒结构蛋白的疫苗开发和抗病毒疗法外，还需要 确定这些黄病毒在细胞内复制周期中的步骤，这些步骤可以作为小分子靶点 分子抑制剂。其中一个步骤是复制病毒RNA，这是一个由依赖病毒RNA的 RNA聚合酶(NSS)，很可能与其他非结构病毒蛋白和 宿主细胞蛋白质。因此，病毒RNA复制复合体的组装提供了独特的蛋白质-蛋白质 一旦确定了这些界面，就可以作为抗病毒治疗的靶点。概述了这些实验 在这项建议中涉及研究登革热病毒和西尼罗河病毒RNA复制的新方法 体外动力学，使用最近开发的高分辨率单分子生物检测技术 是基于交替激光激发(ALEX)荧光光谱。这项创新技术将 随后在检测中实施以筛选黄病毒RNA合成的小分子抑制物。 该项目将(I)开发一台三色激发/四色发射(3cEx/4cEm)ALEX仪器 用于单分子动力学分析的原型；(Ii)开发用于单分子动力学分析的试剂和实验方法 利用哺乳动物和昆虫细胞的细胞质提取物研究黄病毒核糖核酸复制； 确定新型抗病毒药物作为黄病毒RNA复制的特异性抑制剂。总的来说，高分辨率 监控分子相互作用和黄病毒RNA复制动态的能力 提出的分析方法和体外分析方法将极大地帮助结构-活性关系(SAR)研究 采用结构导向的合理药物设计进行先导优化。

Dengue virus and West Nile virus are mosquito-borne flaviviruses with considerable potential as agents for bioterrorism. These positive-strand RNA viruses are also emerging pathogens involved in natural disease outbreaks, making their control a significant public health goal worldwide (especially dengue virus). In addition to vaccine development and anti-viral therapeutics aimed at viral structural proteins, there is a need to identify steps in the intracellular replication cycles of these flaviviruses that can be targeted by small molecule inhibitors. One such step is the replication of viral RNA, a process mediated by the viral RNAdependent RNA polymerase (NSs), most likely in conjunction with other non-structural viral proteins and host cell proteins. Thus, the assembly of viral RNA replication complexes provides unique protein-protein interfaces that once identified, can be utilized as targets for anti-viral therapeutics. The experiments outlined in this proposal involve novel approaches to study dengue virus and West Nile virus RNA replication dynamics in vitro, using a recently-developed high-resolution single molecule bio-detection technology that is based on alternating laser excitation (ALEX) fluorescence spectroscopy. This innovative technology will subsequently be implemented in assays to screen for small molecule inhibitors of flavivirus RNA synthesis. The proposed project will (i) develop a 3-color Excitation/4-color Emission (3cEx/4cEm) ALEX instrument prototype for analysis of single molecule dynamics; (ii) develop reagents and experimental approaches for in uifro flavivirus RNA replication studies using cytoplasmic extracts from mammalian and insect cells; (iii) identify novel anti-viral agents as specific inhibitors of flavivirus RNA replication. Overall, the highresolution power to monitor molecular interactions and flavivirus RNA replication dynamics using the proposed analytical methods and in vitro assays will greatly aid SAR (structure-activity relationship) studies for lead optimization using structure-guided rational drug design.