STRUCTURE OF VIRAL RNA AND ITS ROLE IN ASSEMBLY

描述（申请人提供）：各种物理和化学技术，其中主要是X射线晶体学，将被应用于一系列二十面体病毒，其最终目标是（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，以及（3）设计与现有分子生物学数据一致的病毒体组装和拆卸模型。待研究的病毒是卫星烟草花叶病毒（STMV）、卫星烟草坏死病毒（STNV）、卫星黍花叶病毒（SPMV）、芜菁黄花叶病毒（TYMV）、山蚂蝗黄斑驳病毒（DYMV）、番茄花叶病毒（TAV）、雀麦花叶病毒（BMV）、黍花叶病毒（PMV）和后五种中每一种的空衣壳。这些病毒的结构最为人所知，RNA在病毒体中至少部分可见，并且存在最多的经验。将通过消化完整病毒制备核心RNA颗粒，并将其结晶用于X射线衍射分析。空衣壳将被制造，结晶，并且它们的衍射数据用于不同的傅立叶合成以揭示RNA结构。随着所提出的化学和酶探针信息的整合，以及来自计算机建模的预测以补充X射线研究，将出现结构与功能的全面和解。我们已经证明，当原子力显微镜与化学和酶试剂结合使用时，可以解剖复杂的病毒，以揭示其内部成分，包括核酸。为了扩大我们的调查核酸构象和包装更大，更复杂的病毒，我们将扩大我们的AFM调查的内部结构的艾滋病毒，单纯疱疹病毒，小鼠白血病病毒，和牛痘病毒。这项研究意义重大，因为目前还不知道病毒核酸组分的构象，也没有任何全面的结构基础来描述病毒的组装和分解过程。

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.