New approach to study West Nile virus fusion into cells

研究西尼罗河病毒融入细胞的新方法

• 批准号: 7267936
• 负责人: ILA R SINGH
• 金额: $ 19.54万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7267936
• 关键词: Adopted; Alanine; Amino Acid Sequence; Binding; Biological Assay; Birds; Cell fusion; Cells; Cessation of life; Data; Drug usage; Epidemic; Evaluation; Facility Construction Funding Category; Flavivirus; Flow Cytometry; Generations; Genetic; Glycoproteins; Goals; Green Fluorescent Proteins; Hepatitis C virus; Individual; Libraries; Licensing; Maps; Mediating; Methods; Mutagenesis; Mutation; Nervous System Trauma; North America; Nucleic Acids; Nucleotides; Pathway interactions; Peptide Receptor; Peptide Sequence Determination; Process; Proteins; Public Health; Reporter; Role; Role playing therapy; Screening procedure; Site-Directed Mutagenesis; Sorting - Cell Movement; Testing; Vaccines; Viral; Virus; Virus Diseases; West Nile virus; base; design; env Gene Products; expression cloning; genetic analysis; inhibitor/antagonist; interest; member; mutant; novel; novel strategies; protein function; receptor; receptor binding; research study; small molecule; virus envelope

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.

描述（由申请方提供）：本提案概述了研究西尼罗河病毒包膜蛋白在病毒与细胞融合中所起作用的实验。我们提出了一个大规模的基因和功能分析的包膜蛋白使用我们的新方法的遗传足迹，我们已经开发并成功地应用于其他病毒的方法。与大多数常规的诱变方法不同，这些方法涉及单独分离、储存和表征每个突变体，遗传足迹法允许高效构建和并行分析数千个突变。我们的目标是确定这些蛋白质中对病毒进入宿主细胞至关重要的结构域。如此详细的功能图谱将使我们开始获得对病毒融合的机制性理解。目的1包括使用基于转座子的诱变方法在西尼罗病毒的E和prM/M包膜蛋白中创建全面的、接近饱和的突变库。目的2包括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的区域。目标3包括分析包膜蛋白中的单个突变，以详细了解病毒进入的机制。我们目前的数据显示，成功地产生了一个库的插入突变的感染性克隆西尼罗河病毒，其中包含一个荧光标记。我们还提出了新的细胞-细胞融合试验的细节，我们已经建立了使用丙型肝炎病毒（另一种黄病毒）包膜蛋白，作为初步数据。将为WNV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。这些信息将有助于产生更好的疫苗和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，造成一些感染者的神经损伤和死亡。这一流行病的性质继续发生变化，人们仍然不了解它。由于缺乏特异性治疗方法或许可的疫苗，因此我们了解病毒感染宿主细胞的基本过程尤为重要。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断病毒感染。