Center for Structural Biology of HIV RNA

HIV RNA结构生物学中心

• 批准号: 10641988
• 负责人: ALICE TELESNITSKY
• 金额: $ 81.51万
• 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/10641988
• 关键词: 5' Untranslated Regions; 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; 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; 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.

该项目包括对受感染细胞、病毒和病毒中 HIV-1 RNA 上形成的结构进行深入分析。 与其相互作用的宿主蛋白，以及这些结构在 HIV-1 基因表达中发挥的关键作用。这 病毒使用特定的RNA结构来调节基因表达，但在许多情况下实际的折叠结构 RNA 以及它们与蛋白质形成的更大复合物的细节尚不清楚。我们有 组建了一支强大的调查人员团队，拥有确定问题所需的广泛技能和专业知识 结构，定义生物活性所需的 RNA 的基本部分，并解开 促进病毒基因表达和复制的RNA-蛋白质复合物的作用机制。这 我们建议检查的病毒生命周期的步骤将包括： RNA 合成 RNA 转录本 聚合酶 II 延伸，由 Tat 蛋白、P-TEFb 亚基介导的停滞释放所允许 和 TAR RNA； HIV-1 RNA 的选择性剪接，由剪接位点的顺式作用元件控制 病毒前体RNA；剪接、部分剪接和未剪接 mRNA 的选择性核输出 由 RRE 元件的 Rev 作用和核孔亚基控制；以及病毒 mRNA 的翻译， 包括 5' 帽在决定 RNA 命运中的作用，以及该位点 RNA 发夹的活性 翻译移码在调节长 Gag-Pro-Pol 前体蛋白表达中的作用。我们将学习 溶液中“裸露”RNA 的结构，以及 RNA-蛋白质复合物的结构，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素 参与这些不同的过程、诱变和基于 CRISPR 的敲除，以探究这些过程的功能 因素及其与 RNA 相互作用的细节。我们已经认识到许多 RNA 结构是高度动态的，由一系列替代形式组成——在以下情况下显然是这样的： TAR 元件、控制 RNA 利用的 5' 帽和 5' UTR 序列、剪接调节元件以及 平移移码元素。我们的团队将应用能够监控这些动态的先进方法 多个时间尺度的重新排列。结果将在发现、设计和优化方面开辟新天地 针对RNA的病毒抑制剂。我们首先将解决所有这些 RNA 结构及其功能 T 细胞裂解性生长中病毒活跃复制的背景，很容易在培养物中进行研究。此外，我们还 对建立和维持潜伏期时发生的这些步骤的独特调节感兴趣， 允许病毒作为转录沉默原病毒持续存在的状态。我们将利用新模式 潜伏期来定义 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。该项目的多名研究者经验丰富，高度关注问题，并且 准备在项目概述的每个目标上取得快速进展。