STRUCTURE OF VIRAL RNA AND ITS ROLE IN ASSEMBLY

描述（由申请人提供）：多种物理和化学技术，其中主要是x射线晶体学，将应用于一系列二十面体病毒，最终目的是：(1)推断病毒内部RNA基因组的详细结构，(2)确定重要的蛋白质-核酸相互作用，(3)设计符合现有分子生物学数据的病毒粒子组装和拆卸模型。待研究的病毒包括卫星烟草花叶病毒（STMV）、卫星烟草坏死病毒（STNV）、卫星烟叶花叶病毒（SPMV）、萝卜黄花叶病毒（TYMV）、葡萄黄花叶病毒（DYMV）、番茄曲霉病毒（TAV）、雀麦花叶病毒（BMV）、烟叶花叶病毒（PMV），以及后五种烟叶的空衣壳。这些病毒的结构最为人所知，它们的RNA至少在病毒粒子中部分可见，而且它们的经验最多。核心RNA颗粒将通过消化完整的病毒来制备，并将结晶用于x射线衍射分析。空衣壳将被制造、结晶，它们的衍射数据将被用于差分傅立叶合成来揭示RNA的结构。结合提出的化学和酶探针信息，以及计算机建模预测来补充x射线研究，将出现结构与功能的全面协调。我们已经证明，当原子力显微镜与化学和酶试剂一起应用时，可以解剖复杂的病毒，以揭示包括核酸在内的内部成分。为了将我们对核酸构象和包装的研究扩展到更大、更复杂的病毒，我们将扩展我们对HIV、单纯疱疹病毒、小鼠白血病病毒和牛痘病毒内部结构的AFM研究。这项研究意义重大，因为目前尚不知道病毒的核酸成分的构象，也没有病毒组装和拆卸过程的全面结构基础。

DESCRIPTION (provided by applicant): A variety of physical and chemical techniques, chief among them being X-ray crystallography, will be applied to a series of icosahedral viruses with the ultimate objectives of (1) deducing the detailed structure of the RNA genomes inside the viruses, (2) identifying the important protein-nucleic acid interactions, and (3) devising models for the assembly and disassembly of the virions consistent with existing molecular biological data. Viruses to be studied are Satellite Tobacco Mosaic Virus (STMV), Satellite Tobacco Necrosis Virus (STNV), Satellite Panicum Mosaic Virus (SPMV), Turnip Yellow Mosaic Virus (TYMV), Desmodium Yellow Mottle Virus (DYMV), Tomato Aspermy Virus (TAV), Brome Mosaic Virus (BMV), Panicum Mosaic Virus (PMV), and empty capsids of each of the last five species. These are viruses whose structures are best known, for which RNA is at least partially visible in the virion, and for which the greatest amount of experience exists. Core RNA particles will be prepared by digestion of the intact viruses and will be crystallized for X-ray diffraction analyses. Empty capsids will be made, crystallized, and their diffraction data used in difference Fourier syntheses to reveal RNA structure. With integration of the proposed chemical and enzymatic probe information, and predictions from computer modeling to supplement the X-ray studies, a comprehensive reconciliation of structure with function will emerge. We have shown that when atomic force microscopy is applied in conjunction with chemical and enzymatic reagents, complex viruses can be dissected to reveal internal components, including nucleic acid. To extend our investigations of nucleic acid conformation and packaging to larger, more complex viruses we will extend our AFM investigations of the internal structures of HIV, herpes simplex virus, mouse leukemic virus, and vaccinia virus. This research is significant because the conformation of the nucleic acid component of no virus is currently known, nor is there any comprehensive structural basis for the processes of viral assembly and disassembly.