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.

该项目包括深入分析感染细胞中HIV-1 RNA的结构，病毒和 与它们相互作用的宿主蛋白，以及这些结构在HIV-1基因表达中发挥的关键作用。的 病毒使用特定的RNA结构来调节基因表达，但在许多情况下， RNA和它们与蛋白质形成的更大的复合物的任何细节都还不清楚。我们有 组建了一个强大的调查团队，他们拥有广泛的技能和专业知识， 结构，以确定生物活性所需的RNA的基本部分，并解开 RNA-蛋白质复合物促进病毒基因表达和复制的作用机制。的 我们打算研究的病毒生命周期的步骤包括：RNA转录物的合成 聚合酶II延伸，如通过从由达特蛋白介导的停滞释放所允许的，P-TEFb亚基 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的选择性核输出的剪接，部分剪接和未剪接的mRNA， 由RRE元件处的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RNA发夹在该位点的活性， 翻译移码调节长Gag-Pro-Pol前体蛋白的表达。我们将研究 在溶液中的“裸”RNA的结构，但也在RNA-蛋白质复合物的情况下，因为它们存在于完整的 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 参与这些不同过程的基因，诱变和基于CRISPR的敲除，以探测这些基因的功能。 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-在 TAR元件、控制RNA利用的5'帽和5' UTR序列、剪接调控元件，以及 翻译移码元件我们的团队将采用先进的方法，能够监测这些动态 在多个时间尺度上的重排。这些结果将在发现、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结构和它们的功能， 在T细胞中裂解性生长的活跃复制病毒的背景下，容易在培养物中研究。此外，我们在 感兴趣的是在建立和维持潜伏期时发生的这些步骤的独特调节， 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 的潜伏期来定义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细胞本项目中的多名研究人员经验丰富，高度关注问题， 准备在项目概述的每个目标上取得快速进展。