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.

摘要 基于20年前的突变研究，HIV-1整合酶（IN）蛋白被认为在HIV-1的基因突变中起作用。 在HIV-1复制的晚期阶段中的作用。这些突变统称为II类突变， 不利地影响病毒复制的多个步骤，包括颗粒组装、成熟和随后的 逆转录一些II类突变特异性地损害颗粒成熟，从而导致 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 锥形衣壳核心。然而，近20年来，关于IN如何为 病毒颗粒的成熟 变构IN抑制剂（ALLINI）是近年来出现的一类很有前途的新型抗逆转录病毒药物 一些化合物目前正在进行临床试验。虽然ALLINI最初被设计为阻止 IN与其细胞辅因子LEDGF/p75的相互作用，最近已经表明，它们有效地损害了细胞内的细胞因子。 HIV-1复制的后期步骤。与某些II类IN突变相似，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 已经显示ALLINI可促进异常IN多聚化，该事件如何不利地导致 RNP在衣壳核心外的错误定位仍然未知。 根据这些观察，我们最近探讨了 有趣的可能性是，IN可能与病毒结合， 成熟颗粒中的RNA基因组，ALLINI可能会干扰对正常细胞生长至关重要的IN-RNA相互作用。 粒子形成 为了验证这一假设，我们采用了尖端的CLIP-seq（交联- 免疫沉淀-测序）方法和互补的生物化学方法。这些研究 首次揭示了IN与病毒RNA基因组上的特定序列结合， IN和ALLINI内的突变有效地阻断这些相互作用。我们建议继续我们的研究， 确定ALLINI影响IN-RNA相互作用的机制以及IN-RNA如何影响IN-RNA相互作用的机制的细节。 相互作用调节颗粒成熟。我们还建议使用异常形成的偏心芯 在存在ALLINI/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 研究以了解HIV-1复制的早期入境后事件。