REGULATION AND TARGETING OF HIV-1 INTEGRASE-RNA INTERACTIONS

HIV-1 整合酶-RNA 相互作用的调控和靶向

• 批准号: 9271492
• 负责人: Sebla B. Kutluay
• 金额: $ 30.12万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9271492
• 关键词: Adverse event; Affect; Anti-Retroviral Agents; Binding; Biochemical; Biological Assay; Capsid; Cells; Clinical; Clinical Trials; Complementary DNA; Complex; Databases; Defect; Development; Drug resistance; Electron Microscopy; Elements; Enzymes; Event; Genome; Goals; HIV-1; HIV-1 integrase; Impairment; Infection; Integrase; Integrase Inhibitors; Lead; Methodology; Molecular; Molecular Biology; Morphology; Mutation; Pharmaceutical Preparations; Play; Positioning Attribute; Process; Property; Proteins; RNA; RNA Binding; RNA-Directed DNA Polymerase; Regulation; Research; Resistance; Reverse Transcription; Ribonucleoproteins; Role; Structure; TRIM5 gene; Testing; Time; Vaccines; Variant; Viral; Viral Genome; Viral Vector; Virion; Virus Integration; Virus Replication; Virus-like particle; base; cofactor; crosslinking and immunoprecipitation sequencing; design; inhibitor/antagonist; innovation; insight; mutant; novel; particle; prevent; prototype; pyridine; quinoline; tool; transcriptional coactivator p75; viral RNA; virus morphology

Abstract Based on mutational studies two decades ago, HIV-1 Integrase (IN) protein has been proposed to play a role in late stages of HIV-1 replication. These mutations, collectively referred to as Class II mutations, adversely affect multiple steps of virus replication including particle assembly, maturation and subsequent reverse transcription. Some class II mutations specifically impair particle maturation leading to the formation of aberrant viral cores in which the viral ribonucleoprotein complexes (RNPs) are mislocalized outside of the conical capsid core. However, for nearly 20 years it has remained enigmatic as to how IN can contribute to proper viral particle maturation. Allosteric IN inhibitors (ALLINIs) have recently emerged as a promising new class of antiretroviral agents and select compounds are currently in clinical trials. Although ALLINIs were initially designed to block the interaction of IN with its cellular cofactor LEDGF/p75, it has recently been shown that they potently impair the late steps of HIV-1 replication. Similar to certain Class II IN mutations, these inhibitors selectively interfere with proper virus particle maturation and yield non-infectious particles with eccentrically positioned RNPs. Although it has been shown that ALLINIs can promote aberrant IN multimerization, how this event adversely results in the mislocalization of the RNPs outside the capsid core has remained unknown. Based on these observations, we have recently explored the intriguing possibility that IN may bind the viral RNA genome in mature particles and that ALLINIs may interfere with IN-RNA interactions critical for proper particle formation. To test this hypothesis, we have employed the cutting-edge CLIP-seq (crosslinking- immunoprecipitation-sequencing) methodology and complementary biochemical approaches. These studies have revealed for the first time that IN binds to specific sequences on the viral RNA genome and that certain mutations within IN and ALLINIs potently block these interactions. We propose to continue our studies in identifying the details of the mechanism by which ALLINIs affect IN-RNA interactions and how IN-RNA interactions regulate particle maturation. We also propose to use the aberrantly formed eccentric cores generated in the presence of ALLINIs/Class II mutations as a tool to understand the early post-entry events in infection. As such, this project will not only provide unprecedented insight into the novel role of HIV-1 IN in particle assembly and the primary mechanism of action of ALLINIs, but will also facilitate the development of studies to understand early post-entry events in HIV-1 replication.

抽象的 基于二十年前的突变研究，HIV-1 整合酶 (IN) 蛋白被提议发挥作用 HIV-1 复制后期的作用。这些突变统称为 II 类突变， 对病毒复制的多个步骤产生不利影响，包括颗粒组装、成熟和随后的 逆转录。一些 II 类突变特别损害颗粒成熟，导致形成 异常的病毒核心，其中病毒核糖核蛋白复合物 (RNP) 错误定位在病毒核心之外 圆锥形衣壳核心。然而，近 20 年来，IN 如何为社会做出贡献一直是个谜。 适当的病毒颗粒成熟。 变构 IN 抑制剂 (ALLINI) 最近成为一类有前景的新型抗逆转录病毒药物 某些化合物目前正在进行临床试验。尽管 ALLINI 最初的设计目的是阻止 IN 与其细胞辅助因子 LEDGF/p75 的相互作用，最近表明它们会有效损害 HIV-1复制的后期步骤。与某些 II 类 IN 突变类似，这些抑制剂选择性干扰 适当的病毒颗粒成熟并产生具有偏心定位的 RNP 的非感染性颗粒。虽然 研究表明，ALLINI 可以促进异常的 IN 多聚化，该事件如何产生不利结果 RNP 在衣壳核心之外的错误定位仍然未知。 基于这些观察，我们最近探索了 IN 可能与病毒结合的有趣可能性 成熟颗粒中的 RNA 基因组和 ALLINI 可能会干扰 IN-RNA 相互作用，这对正常颗粒至关重要 颗粒形成。 为了检验这一假设，我们采用了尖端的 CLIP-seq（交联- 免疫沉淀-测序）方法和补充生化方法。这些研究 首次揭示 IN 与病毒 RNA 基因组上的特定序列结合，并且某些 IN 和 ALLINI 内的突变可有效阻断这些相互作用。我们建议继续学习 确定 ALLINI 影响 IN-RNA 相互作用的机制细节以及 IN-RNA 如何 相互作用调节粒子的成熟。我们还建议使用异常成型的偏心铁芯 在 ALLINI/II 类突变存在的情况下生成，作为了解早期进入后事件的工具 感染。因此，该项目不仅将为 HIV-1 IN 的新作用提供前所未有的见解 颗粒组装和 ALLINI 的主要作用机制，但也将促进 研究了解 HIV-1 复制的早期进入后事件。