Structure of the HIV-1 Genome

 描述（由申请人提供）：该项目的重点是生物相关环境中RNA分子三维结构的表征，广泛联系生物医学科学中的重要问题。首先，人类免疫缺陷病毒（艾滋病毒）目前感染全球3 500万人，每年约有300万新感染者。对病毒蛋白质成分的研究已导致开发出多种有效药物，但还需要其他有效的抗逆转录病毒药物。原则上，RNA基因组本身就是一个引人注目的药物靶点。我们已经确定的HIV-1基因组RNA中的保守功能基序-以及我们将作为本提案的一部分表征的元素-作为抗逆转录病毒药物开发的新靶点和途径具有重要的前景。其次，HIV-1 RNA基因组的高级结构中编码的信息密度非常大，这是遗传密码的关键组成部分，我们目前对它的了解还很有限。这项研究计划将增强我们对RNA介导的基因调控原理的理解。它还将促进我们对RNA病毒基因组的组织和压缩的理解，并将阐明适用于密集组织RNA的原则，包括lncRNA，核体和其他调控元件。第三，由我们实验室开创的SHAPE探测策略产生定量但高度特异性的核苷酸分辨率信息，因此，我们现在专注于开发多种，替代，互补的策略，专门检测RNA中真正的空间和高阶相互作用。最后，在整个项目中，我们将创建综合策略，将多个，互补的，实验简洁的，物理上准确的结构探测技术与用户友好的数据处理相结合，使非专家实验室在相关细胞环境中对大RNA进行简单和全面的功能分析成为可能。我们的合作团队由HIV病毒学家、RNA结构生物学家和 生物信息学专家，将解决以下目标：（1）使用基因组规模的SHAPE-MaP策略分析真实病毒体内完整HIV-1基因组的结构;（2）通过大规模，单分子，相关化学探测实验确定整个HIV-1基因组的空间相互作用;（3）全面定义介导包装的HIV-1基因组RNA的RNA相互作用组的分子内和分子间相互作用;以及（4）创建强大的平台无关软件，用于专门测量RNA中的空间相互作用的化学探测实验的全自动分析。

 DESCRIPTION (provided by applicant): This project, focused on characterization of the three-dimensional structures of RNA molecules in biologically relevant environments, links broadly important problems in biomedical science. First, the human immunodeficiency virus (HIV) currently infects 35 million individuals globally with roughly 3 million new infections per year. Studies of the protein components of the virus have led to the development of multiple effective drugs, but additional effective antiretroviral agents are needed. In principle, the RNA genome itself is a compelling drug target. The conserved, functional motifs in the HIV-1 genomic RNA that we have identified - and elements that we will characterize as part of this proposal - have significant promise as novel targets and pathways for antiretroviral drug development. Second, the extraordinary density of information encoded in the higher-order structure of the HIV-1 RNA genome represents a key component of the genetic code, one that we understand poorly at present. This research program will enhance our understanding of the principles that govern RNA-mediated gene regulation. It will also advance our understanding of the organization and compaction of RNA virus genomes and will illuminate principles that apply to densely organized RNAs including lncRNAs, nuclear bodies, and other regulatory elements. Third, the SHAPE probing strategy, pioneered by our lab, yields quantitative but highly specific nucleotide-resolution information, and, therefore, we are now focusing on developing multiple, alternative, complementary strategies that specifically detect true through-space and higher-order interactions in RNA. Finally, throughout this project, we will create integrated strategies that meld multiple, complementary, experimentally concise, and physically accurate structure probing technologies with user-friendly data processing to make possible facile and comprehensive functional analyses of large RNAs in relevant cellular contexts by non-expert laboratories. Our collaborative team, consisting of HIV virologists, RNA structural biologists, and experts in bioinformatics, will tackle the following Aims: (1) Analyze the structure of complete HIV-1 genomes inside authentic virions using the genome-scale SHAPE-MaP strategy; (2) identify through-space interactions across the entire HIV-1 genome by large-scale, single-molecule, correlated chemical probing experiments; (3) comprehensively define the intra- and inter-molecular interactions that mediate the RNA interactome of packaged HIV-1 genomic RNAs; and (4) create robust platform-independent software for fully automated analysis of chemical probing experiments that specifically measure through-space interactions in RNA.