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: 10062475
• 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/10062475
• 关键词: Adverse event; Affect; Anti-Retroviral Agents; Antiviral Therapy; Binding; Biochemical; Biological Assay; Capsid; Cells; Clinical; Clinical Trials; Complementary DNA; Complex; Data; Defect; Development; Drug resistance; Electron Microscopy; Elements; Enzymes; Event; Genome; Goals; HIV-1; Human Activities; 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 根据两十年前的突变研究，已经提出HIV-1整合酶（IN）蛋白来发挥 在HIV-1复制的后期阶段的作用。这些突变，共同称为II类突变， 不利地影响病毒复制的多个步骤，包括颗粒组装，成熟和随后 逆转录。一些II类突变特异性损害了粒子成熟，导致形成 病毒核糖核蛋白复合物（RNP）的异常病毒核被错误定位在 圆锥形衣壳芯。但是，在近20年的时间里 适当的病毒颗粒成熟。 变构抑制剂（Allinis）最近已成为一种有希望的新类抗逆转录病毒药物 当前，精选的化合物正在临床试验中。尽管最初设计了Allinis，以阻止 IN与其细胞辅助因子LEDGF/p75的相互作用，最近证明它们有效损害了 HIV-1复制的后期步骤。与突变中的某些II类相似，这些抑制剂有选择地干扰 适当的病毒颗粒成熟并产生偏心定位的RNP的非感染颗粒。虽然 已经表明，阿尼尼斯在多聚化方面可能促进异常，这一事件如何不利地导致 Capsid核心以外的RNP的错误定位尚不清楚。 基于这些观察，我们最近探索了 在可能结合病毒的可能性很大的可能性 成熟颗粒中的RNA基因组，Allinis可能会干扰适当至关重要的RNA相互作用 粒子形成。 为了检验这一假设，我们采用了尖端的夹子序列（交联 - 免疫沉淀 - 测序）方法和互补的生化方法。这些研究 首次揭示了与病毒RNA基因组上的特定序列结合的，并且 内部和Allinis内部的突变会有力阻止这些相互作用。我们建议继续学习 识别Allinis影响RNA相互作用的机制的细节以及RNA如何 相互作用调节粒子成熟。我们还建议使用异常形成的偏心核 在存在Allinis/II类突变的情况下生成，作为了解早期进入后事件的工具 感染。因此，该项目不仅将对HIV-1在IN中的新作用提供前所未有的见解 粒子组装和阿里尼斯的主要作用机理，但也将促进 了解HIV-1复制中早期进入后事件的研究。

项目成果

CLIP-related methodologies and their application to retrovirology.

• DOI: 10.1186/s12977-018-0417-2
• 发表时间: 2018-05-02
• 期刊: Retrovirology
• 影响因子: 3.3
• 作者: Bieniasz PD;Kutluay SB
• 通讯作者: Kutluay SB