ENHANCED MICROSCOPY AND ANALYSIS OF VIRUSES

本提案的目标是显著改进 成像、重建和解释三维结构 的二十面体病毒，并将这种方法应用于一些 与病毒结构和功能相关的重要问题， 与Parti的项目同事合作。 的主要工具 冷冻电镜和图像重建。 这些方法目前允许三维结构信息， 在20-30埃分辨率水平下获得大分子 保持在水环境中的标本， “自然”（生理）条件。 增加了高分辨率的慢- 扫描、电荷耦合器件（CCD）照相机和相关外围设备， 计算机设备到透射电子显微镜， 开发计算机软件，以捕获和分析数字，低， 冷冻水化样品的点扫描辐照图像 将大大提高研究病毒和病毒复合体的能力， 分辨率超过20埃。 几种病毒的结构以及病毒抗体和病毒 受体复合物将用新技术进行研究。 这些研究 包括检查：甲病毒（以研究i）构象 由各种物理处理诱导的病毒包膜的变化，ii） 与单克隆抗体和细胞受体的相互作用，和iii） 重组核心颗粒）;豇豆花叶病毒的嵌合体， 包括来自人鼻病毒长度为15-22个残基的多肽环 血清型14和来自人gp 41和gp 120蛋白的环 免疫缺陷病毒;人鼻病毒和中和复合物 抗体和细胞间粘附分子ICAM-1; 犬和人细小病毒的单克隆抗体; 同源噬菌体phiX 174、G4和α 3。

The goals of this proposal are to markedly improve the methodology for imaging, reconstructing, and interpreting the three-dimensional structures of icosahedral viruses and to apply this methodology to a number of significant questions related to viral structure and function in collaboration with program-project colleagues at Parti. The primary tools of investigation are cryo-electron microscopy and image reconstruction. These methods currently allow three-dimensional structural information to be obtained at the 20-30 angstroms resolution level for macromolecular specimens maintained in an aqueous environment that closely mimics 'native' (physiological) conditions. Addition of a high resolution slow- scan, charge-coupled device (CCD) camera and associated peripherals and computer equipment to a transmission electron microscope and the development of computer software to capture and analyze digital, low- irradiation images of frozen-hydrated specimens with spot scan procedures will greatly improve the ability to study viruses and virus complexes at resolutions exceeding 20 angstroms. The structures of several viruses and also virus-antibody and virus- receptor complexes will be studied with the new technology. These studies include examinations of: alphaviruses (to investigate i) conformational changes in the virus envelope induced by various physical treatments, ii) interactions with monoclonal antibodies and cellular receptors, and iii) reconstituted core particles); chimeras of cowpea mosaic virus which include polypeptide loops, 15-22 residues in length, from human rhinovirus serotype 14, and loops from proteins gp41 and gp120 of human immunodeficiency virus; complexes of human rhino virus and neutralizing antibodies and the intercellular adhesion molecule, ICAM-1; complexes of canine and human parvoviruses with monoclonal antibodies; and proheads of homologous bacteriophages phiX174, G4, and alpha3.