New approach to study West Nile virus fusion into cells

描述（由申请人提供）：本提案概述了研究西尼罗病毒包膜蛋白在病毒与细胞融合过程中所起作用的实验。我们建议使用我们的遗传足迹新方法对包膜蛋白进行大规模的遗传和功能分析，这种方法我们已经开发并成功应用于其他病毒。与大多数传统的诱变方法不同，这些方法涉及分离、存储和表征每个突变，遗传足迹允许对数千个突变进行有效的构建和并行功能分析。我们的目标是确定这些蛋白中对病毒进入宿主细胞至关重要的结构域。这种详细的功能映射将使我们能够开始获得对病毒融合的机制理解。目标1包括使用基于转座子的诱变方法，在西尼罗病毒E和prM/M包膜蛋白中创建全面的、接近饱和的突变文库。目的2包括对突变库的平行功能分析，以确定病毒融合所必需的包膜蛋白区域。目的3包括分析包膜蛋白的个体突变，以获得对病毒进入机制的详细了解。我们目前的数据显示，在一个含有荧光标记的西尼罗河病毒传染性克隆中，成功地产生了一个插入突变文库。我们还介绍了我们使用丙型肝炎病毒（另一种黄病毒）包膜蛋白建立的新型细胞-细胞融合试验的细节，作为初步数据。将为西尼罗河病毒开发类似的融合试验。鉴定融合所必需的结构域将有助于选择进一步分析的区域，并有助于理解病毒进入的过程。这一信息将有助于生产更好的疫苗，以及利用小分子抑制剂靶向融合过程。与公共卫生的相关性：自1999年传入北美以来，西尼罗河病毒已传播到美国大部分地区，造成一些感染者神经损伤和死亡。这一流行病的性质在继续变化，人们仍然不了解它。由于缺乏专门的治疗方法或获得许可的疫苗，我们了解病毒感染宿主细胞的基本过程变得尤为重要。我们对包膜蛋白的全面研究将提供这些信息，最终目标是能够使用药物和疫苗阻止病毒感染。

DESCRIPTION (provided by applicant): This proposal outlines experiments to study the roles played by the West Nile virus envelope proteins in viral fusion with the cell. We propose a large-scale genetic and functional analysis of the envelope proteins using our novel approach of genetic footprinting, a method that we have developed and applied successfully to other viruses. In contrast to most conventional methods of mutagenesis, which involve the isolation, storage and characterization of each mutant separately, genetic footprinting allows for efficient construction and parallel functional analysis of thousands of mutations. Our objective is to identify domains in these proteins that are critical for viral entry into host cells. Such detailed functional mapping will allow us to begin to gain a mechanistic understanding of viral fusion. Aim 1 consists of creating comprehensive, near-saturating libraries of mutations in the E and prM/M envelope proteins of West Nile virus, using a transposon-based mutagenesis method. Aim 2 consists of a parallel functional analysis of the library of mutations to define regions of the envelope proteins that are necessary for viral fusion. Aim 3 consists of analyzing individual mutations in envelope proteins to obtain a detailed understanding of the mechanism of viral entry. We present data to show the successful generation of a library of insertional mutations in an infectious clone of WNV that contains a fluorescent marker. We also present details of novel cell-cell fusion assay that we have set up using hepatitis C virus (another flavivirus) envelope proteins, as preliminary data. A similar fusion assay will be developed for WNV. Identification of domains essential for fusion will help in selecting regions for further analysis, and in understanding the process of viral entry. This information will be useful in generating better vaccines and in targeting the fusion process with small molecule inhibitors. Relevance to Public Health: Since its introduction to North America in 1999, West Nile virus has spread to most of the US, causing neurological damage and death in some infected individuals. The epidemic continues to change in character and is still not understood. Lack of specific treatment or a licensed vaccine make it especially important that we understand the fundamental processes by which the virus infects the host cell. Our comprehensive study of the envelope proteins will provide such information with the eventual goal of being able to block viral infection using drugs and vaccines.