Center for Structural Biology of HIV RNA

HIV RNA结构生物学中心

• 批准号: 10505795
• 负责人: ALICE TELESNITSKY
• 金额: $ 79.7万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-09 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10505795
• 关键词: 5' Untranslated Regions; Address; Alternative Splicing; Binding; Binding Proteins; Biological; Budgets; Cell Nucleus; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA Polymerase II; Dimerization; Elements; Event; Gene Expression; Gene Silencing; Genetic; Genetic Transcription; Growth; Guide RNA; HIV; HIV-1; Head; Human Resources; Integration Host Factors; Intervention; Investigation; Knock-out; Laboratories; Lead; Libraries; Life Cycle Stages; Location; Lytic; Maintenance; Mediating; Messenger RNA; Methods; Modeling; Modification; Monitor; Mutagenesis; Nuclear Export; Nuclear Pore; Phase; Play; Polymerase; Positive Transcriptional Elongation Factor B; Process; Protein Precursors; Proteins; Proteome; Proviruses; RNA; RNA Binding; RNA Polymerase II; RNA Precursors; RNA Probes; RNA Splicing; RNA chemical synthesis; Regulation; Regulatory Element; Research Personnel; Ribosomal Frameshifting; Role; Site; Structure; T memory cell; T-Lymphocyte; Technology; Time; Transcript; Transcription Elongation; Transcriptional Regulation; Translations; Viral; Viral Genes; Virus; Virus Inhibitors; Virus Replication; Work; base; cis acting element; design; experience; interest; mRNA Export; member; novel; protein complex; screening; skills; small molecule; structural biology; tat Protein; tool; trafficking; viral DNA; viral RNA

This Project consists of a deep analysis of the structures that form on HIV-1 RNAs in infected cells, the viral and host proteins with which they interact, and the critical roles these structures play in HIV-1 gene expression. The virus uses specific RNA structures to regulate gene expression, but in many cases the actual folded structures of the RNAs and of the larger complexes they form with proteins are not yet known in any detail. We have assembled a powerful team of investigators with the broad range of skills and expertise needed to determine the structures, to define the essential portions of the RNAs needed for biological activity, and to unravel the mechanism of action of the RNA-protein complexes that promote virus gene expression and replication. The steps of the viral life cycle that we propose to examine will include: the synthesis of RNA transcripts by RNA polymerase II elongation, as allowed by the release from arrest mediated by the Tat protein, P-TEFb subunits and the TAR RNA; the alternative splicing of HIV-1 RNAs, as controlled by cis-acting elements at the splice sites of the viral precursor RNA; the selective nuclear export of spliced, partially spliced and unspliced mRNAs as controlled by Rev action at the RRE element and by nuclear pore subunits; and the translation of viral mRNAs, including the role of the 5' cap in determining the fate of the RNAs, and the activity of the RNA hairpin at the site of translational frameshifting in regulating expression of the long Gag-Pro-Pol precursor protein. We will study structures of “naked” RNAs in solution, but also in the context of RNA-protein complexes as they exist in intact infected cells. We will use powerful genetic tools and rapid readouts of gene expression to identify host factors involved in these various processes, mutagenesis and CRISPR-based knockouts to probe the functions of these factors and the details of their interactions with RNA. We have come to appreciate that many of the RNA structures are highly dynamic and consist of a constellation of alternative forms – clearly true in the cases of the TAR element, the 5' cap and 5' UTR sequence that control RNA utilization, the splicing regulatory elements, and the translational frameshift element. Our team will apply advanced methods capable of monitoring these dynamic rearrangements at multiple time scales. The results will break new ground in discovery, design, and optimization of viral inhibitors targeting RNA. We initially will address all these RNA structures and their functions in the context of the actively replicating virus in lytic growth in T cells, readily studied in culture. In addition, we are interested in the distinctive regulation of these steps that occurs in the establishment and maintenance of latency, a state allowing persistence of virus as transcriptionally silent proviruses. We will take advantage of a new model of latency to define changes in the RNA structures and the way they are recognized by the altered proteome of the memory T cell. The multiple investigators in this Project are experienced, highly focused on the issues, and prepared to make rapid progress on each Aim outlined for the Project.

该项目包括对感染细胞中HIV-1 RNA上形成的结构的深入分析，病毒和 它们相互作用的宿主蛋白，以及这些结构在HIV-1基因表达中所起的关键作用。这个 病毒使用特定的RNA结构来调节基因的表达，但在许多情况下，实际的折叠结构 其中的RNA和它们与蛋白质形成的更大的复合体的细节尚不清楚。我们有 组建了一支强大的调查团队，拥有确定 结构，以定义生物学活动所需的RNA的基本部分，并解开 促进病毒基因表达和复制的RNA-蛋白质复合体的作用机制。这个 我们建议研究的病毒生命周期的步骤将包括：由RNA合成RNA转录本 聚合酶II的延长，如由TAT蛋白，P-TEFb亚基介导的从停滞中释放所允许的 和TAR RNA；HIV-1 RNA的选择性剪接，受剪接位点上的顺式作用元件控制 指病毒前体RNA；选择性核输出剪接的、部分剪接的和未剪接的mRNAs 受RRE元件上的REV作用和核孔亚基控制；以及病毒mRNAs的翻译， 包括5‘帽在决定RNA命运中的作用，以及RNA发夹在该位点的活性 翻译移码在调节长的Gag-Pro-Pol前体蛋白表达中的作用。我们会研究 裸露的RNA在溶液中的结构，但也在RNA-蛋白质复合体的背景下，因为它们以完整的形式存在 被感染的细胞。我们将使用强大的遗传工具和基因表达的快速读数来识别宿主因子 参与这些不同的过程，突变和基于CRISPR的敲除，以探索这些功能 因子及其与RNA相互作用的细节。我们已经意识到，许多RNA 结构是高度动态的，由一系列可供选择的形式组成-显然在 TAR元件，控制RNA利用的5‘帽和5’非编码区序列，剪接调节元件，以及 平移框架元素。我们的团队将应用先进的方法来监测这些动态 在多个时间尺度上的重新安排。其结果将在发现、设计和优化方面开辟新的天地 以RNA为靶点的病毒抑制剂。我们首先将讨论所有这些RNA结构及其功能 背景是活跃复制的病毒在T细胞中的裂解生长，很容易在培养中研究。此外，我们正在 对在建立和维持等待时间中发生的这些步骤的独特调节感兴趣， 一种允许病毒作为转录沉默的前病毒持续存在的状态。我们将利用一种新的模式 潜伏期的变化来定义RNA结构的变化以及它们被改变的蛋白质组识别的方式 记忆T细胞。该项目中的多名调查人员经验丰富，高度关注问题，并且 准备在项目概述的每一个目标上取得快速进展。