STRUCTURAL ANALYSIS OF HIV GENOMIC RNA

HIV 基因组 RNA 的结构分析

• 批准号: 2887124
• 负责人: PATRICK N ALLEN
• 金额: $ 27.88万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887124
• 关键词: conformation; gene mutation; genetic strain; human immunodeficiency virus 1; nucleic acid sequence; nucleic acid structure; pancreatic ribonuclease; polymerase chain reaction; ribonucleoproteins; virion; virus RNA; virus genetics

The higher-order structure of HIV genomic RNA may be important for formation of a mature core. Results from a number of studies suggest that condensation of the core is essential for infectious viruses. The goal is to study and understand, in detail, the structure of the HIV-1 genomic RNA A phylogenetic comparison of the full-length RNAs from various isolates will provide the framework for a secondary structure model of the entire genome. in addition, structure-specific chemicals and enzymatic probes will be used along with primer extension for further characterization of the structure. Nuclease digestion and chemical modification patterns of several HIV isolates will be compared to identify the conserved structural features of the RNA. Folding properties of in vitro transcribed HIV RNA fragments will be extensively studied. A comparison of structure probing data from in vitro transcripts of HIV RNAs that vary in sizes ranging from approximately 200 to 3000 nucleotides, all with mature 5' ends, will be helpful in understanding how the RNA folds. To determine how RNA conformation influences the structure of the capsid core, it will be necessary to study the structure of genomic RNA inside the core. The core is permeable to nucleases and parts of the RNA inside are susceptible to cleavage (J. Coffin, personal communication). Mild digestion of encapsidated RNA will determine if there are RNase hypersensitive sites. Gradual increase in nuclease concentration and/or denaturating condition, will reveal sites that are protected by higher-order RNA structures or by interaction with core protein(s). Regions of RNA that are resistant to high concentrations of nucleases under denaturing conditions may prove to be structurally interesting and functionally important. Results from the digestion experiments will help to determine if the RNA is folded into major domains. If distinct domains exist, structure probing will be used to determine whether or not the in vitro transcribed RNAs, corresponding to these domains, are able to fold into correct structures independently, or if core proteins are needed to aid in folding the RNA. In order to determine sites on the RNA that are in contact with proteins, the structure of the encapsidated RNA will be compared with phenol extracted RNA. Finally, an attempt will be made to identify structural differences in RNAs from viruses harboring mutations that prevent condensation of the core versus condensed-core RNAs.

HIV基因组RNA的高阶结构可能对于 形成成熟的核心。多项研究结果表明 核心的浓缩对于传染性病毒至关重要。目标是 详细研究和了解 HIV-1 基因组 RNA 的结构 不同分离株的全长 RNA 的系统发育比较 将为整个二级结构模型提供框架 基因组。此外，结构特异性化学品和酶探针将 与引物延伸一起使用以进一步表征 结构。几种核酸酶消化和化学修饰模式 将比较 HIV 分离株以确定保守的结构特征 RNA 的。体外转录的HIV RNA片段的折叠特性 将被广泛研究。结构探测数据的比较 HIV RNA 的体外转录物大小各异，范围从 大约 200 至 3000 个核苷酸，全部具有成熟的 5' 末端，将被 有助于理解 RNA 的折叠方式。 确定 RNA 构象如何影响衣壳的结构 核心，有必要研究基因组RNA内部的结构 核。核心对核酸酶是可渗透的，内部的 RNA 部分是 容易分裂（J. Coffin，个人通讯）。温和消化 衣壳化 RNA 的数量将确定是否存在 RNase 过敏位点。 核酸酶浓度和/或变性条件逐渐增加， 将揭示受高阶RNA结构或受保护的位点 与核心蛋白的相互作用。 RNA 区域具有高抗性 变性条件下核酸酶的浓度可能被证明是 结构有趣且功能重要。结果来自 消化实验将有助于确定 RNA 是否折叠成 主要领域。如果存在不同的域，将使用结构探测 确定RNA是否在体外转录，相应的 对于这些域，能够独立折叠成正确的结构， 或者是否需要核心蛋白来帮助折叠 RNA。为了 确定 RNA 上与蛋白质接触的位点、结构 将衣壳化的 RNA 与苯酚提取的 RNA 进行比较。 最后，将尝试鉴定 RNA 的结构差异 来自含有阻止核心凝结的突变的病毒 与浓缩核心 RNA 相比。