ELECTRON MICROSCOPIC ANALYSIS OF GP120/GP160

描述：（改编自申请人摘要）：详细 了解分子的结构和功能 参与HIV-1病毒与 它的靶细胞是了解这种疾病的基础 以及合理设计疫苗和其他 治疗剂和诊断剂。有一种分子可以说是 在整个感染过程中最重要的，即包膜 糖蛋白gp 120仍然是最不好表征的。 这 该提案描述了旨在确定3D HIV-1包膜糖蛋白gp 120及其 前体，gp 160。 这将通过确定其总体 形状并生成表面的3-D表位和反应位点图 的分子。 两种形式的高分辨率电子显微镜将 可用于单独和组合分析包膜糖蛋白 与各种配体和单克隆抗体结合， 表征域组织和三维表面图 表位和反应位点。负染色电子显微镜将 产生分子和免疫复合物的图像，因为它们粘附在一个 碳基质 这是一种成熟的分析技术， 大分子的结构，虽然它很少用于详细的 分析小至GP 120的分子。 第二种方法将是 使用冷冻电子显微镜观察未染色的分子 悬浮在冰中。 对这些小分子的分析可能是在 这项技术的尖端。 将广泛使用 以前定义和充分表征的试剂。 所产生的信息可以：1）揭示整体域 构型，2）给出分子内（节段） 灵活性，和3）揭示免疫学的拓扑位置， 功能性和序列限定的反应位点和表位。 与此 信息，可以提出更详细的机制， 解释包膜糖蛋白的功能和 有些抗体能中和HIV-1而其他抗体不能。

DESCRIPTION: (Adapted from applicant's abstract): A detailed understanding of the structural and functional aspects of the molecules involved in the interaction, binding, and fusion of the HIV-1 virus with its target cells is fundamental to an understanding of the disease process in AIDS and to the rational design of vaccines and other therapeutic and diagnostic agents. The one molecule that is arguably the most important in the entire infectious process, namely the envelope glycoprotein, gpl20, remains the least well characterized. This proposal describes investigations aimed at determining the 3-D morphology of the HIV-1 envelope glycoproteins, gpl20, and its precursor, gpl60. This will be accomplished by determining its overall shape and generating a 3-D epitope and reactive site map of the surface of the molecule. Two forms of high resolution electron microscopy will be used to analyze the envelope glycoproteins alone and in combination with various ligands and monoclonal antibodies to give a characterization of domain organization and a 3-D surface map of epitopes and reactive sites. Negative stain electron microscopy will yield images of molecules and immune complexes as they adhere to a carbon substrate. This is a well established technique for analyzing the structure of macromolecules though it is rarely used for detailed analysis of molecules as small as gpl20. The second approach will be the use of cryo-electron microscopy where unstained molecules are viewed suspended in ice. Analysis of such small molecules may be at the cutting edge of this technology. Extensive use will be made of previously defined and well characterized reagents. The information generated may: 1) reveal the overall domain configuration, 2) give an indication of the intramolecular (segmental) flexibility, and 3) reveal the topological locations of immunological, functional, and sequence-defined reactive sites and epitopes. With this information, it may be possible to propose more detailed mechanistic explanations for envelope glycoprotein function and for the ability of some antibodies but not others to neutralize HIV-1.