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: 10402641
• 负责人: Sebla B. Kutluay
• 金额: $ 47.25万
• 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 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10402641
• 关键词: Anti-Retroviral Agents; Antiviral Agents; Binding; Biochemical; Biological Assay; C-terminal; CRISPR screen; Capsid; Cells; Clinical; Complement; Complex; Data; Defect; Dependence; Development; Distal; Drug resistance; Electron Microscopy; Electrostatics; Ensure; Enzymes; Evolution; Funding; Generations; Genome; Goals; Guide RNA; HIV-1; Highly Active Antiretroviral Therapy; Human Activities; In Vitro; Innate Immune Response; Integrase; Integrase Inhibitors; Kinetics; Knowledge; Life Cycle Stages; Location; Mediating; Molecular; Morphogenesis; Mutagenesis; Mutation; Nature; Nucleic Acids; Nucleocapsid; Outcome; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Play; Publishing; RNA; RNA Binding; RNA Degradation; RNA Recognition Motif; RNA-Directed DNA Polymerase; Regulation; Reporting; Resistance; Reverse Transcription; Role; Sampling; Small Interfering RNA; Specificity; Testing; Therapeutic; Vaccines; Variant; Viral; Viral Genome; Virion; Virus; Virus Replication; Work; base; experimental study; inhibitor; mutant; novel; particle; physical separation; premature; therapeutic development; therapeutic target; tool; viral RNA

Abstract The emergence of drug resistant human immunodeficiency virus type-1 (HIV-1) variants and the lack of an effective vaccine require the development of novel anti-retroviral drugs. The catalytic activity of HIV-1 integrase (IN) has been successfully targeted by several highly effective and well tolerated IN strand transfer inhibitors (INSTIs). However, despite high barriers with the second-generation INSTIs, mutations conferring resistance to multiple INSTIs have been reported in clinical settings. Thus, targeting IN through an alternative mechanism can complement the existing therapeutic strategies and substantially increase the barrier to emergence of drug resistant HIV-1 variants upon INSTI treatment. IN has long been known to have an enigmatic non-catalytic function in the HIV-1 life cycle. Certain mutations in IN, collectively referred to as class II mutations, are reportedly pleiotropic and result in defects in viral particle assembly, maturation and reverse transcription. In a paradigm-shifting study, we have discovered that HIV-1 IN binds to the viral RNA genome (gRNA) in virions and that this interaction is critically important for accurate virion morphogenesis. Inhibition of IN-gRNA interactions through allosteric integrase inhibitors (ALLINIs) or class II IN substitutions results in the formation of aberrant “eccentric” particles with the gRNA is mislocalized between the empty capsid (CA) lattice and the viral envelope. Furthermore, we have shown that IN tetramerization is critical for RNA-binding and that a number of class II IN substitutions located throughout IN inhibit RNA binding through modulation of IN tetramerization. Finally, work from our lab demonstrated that premature degradation of the gRNA and its physical separation from the reverse transcriptase enzyme in virions underlies the reverse transcription defects of eccentric particles in target cells. Importantly, this untimely gRNA degradation is not due to inhibition of IN-gRNA interactions per se, but rather due to loss of protection with the CA lattice, as a similar outcome was observed upon CA destabilization. Together, these studies cemented the role of IN-gRNA interactions in virion maturation and demonstrated the critical importance of the CA lattice in protection of viral nucleic acids in target cells. Based on these novel findings and extensive preliminary data, we propose to elucidate the nature and rules of HIV-1 IN-gRNA interactions, how IN binding to the gRNA mediates proper assembly of the HIV-1 capsid lattice and how infected cells sense and respond to aberrant particles generated upon inhibition of IN-gRNA interactions and destabilization of the CA lattice. These studies will fill a critical gap in our understanding of the critical noncatalytic function of HIV-1 IN in particle maturation and the consequences of inhibiting these interactions. Together, this project will not only enhance our basic knowledge of HIV-1 replication but also aid in the development of novel antivirals that can complement INSTI-based therapies in clinical settings.

抽象的 耐药人类免疫缺陷病毒 1 型 (HIV-1) 变种的出现以及缺乏 有效的疫苗需要开发新型抗逆转录病毒药物。 HIV-1的催化活性 整合酶 (IN) 已成功被多种高效且耐受性良好的 IN 链转移靶向 抑制剂（INSTI）。然而，尽管第二代 INSTI 存在很高的障碍，但突变赋予了 临床环境中已有对多种 INSTI 耐药的报道。因此，通过替代方案瞄准 IN 机制可以补充现有的治疗策略并大大增加治疗的障碍 INSTI 治疗后出现耐药 HIV-1 变异体。 人们早已知道 IN 在 HIV-1 生命周期中具有神秘的非催化功能。肯定 据报道，IN 突变统称为 II 类突变，具有多效性，并导致缺陷 病毒颗粒组装、成熟和逆转录。在一项范式转换研究中，我们发现 HIV-1 IN 与病毒粒子中的病毒 RNA 基因组 (gRNA) 结合，这种相互作用对于 准确的病毒颗粒形态发生。通过变构整合酶抑制剂抑制 IN-gRNA 相互作用 (ALLINI) 或 II 类 IN 取代会导致 gRNA 形成异常的“偏心”颗粒 错误定位在空衣壳 (CA) 晶格和病毒包膜之间。此外，我们还证明了 IN 四聚化对于 RNA 结合至关重要，并且许多 II 类 IN 取代位于整个 IN 通过调节 IN 四聚化来抑制 RNA 结合。最后，我们实验室的工作表明 gRNA 的过早降解及其与病毒粒子中逆转录酶的物理分离 是靶细胞中偏心颗粒逆转录缺陷的基础。重要的是，这种不合时宜的 gRNA 降解并不是由于 IN-gRNA 相互作用本身受到抑制，而是由于失去了对 IN-gRNA 的保护 CA 晶格，在 CA 不稳定时观察到类似的结果。这些研究共同巩固了 IN-gRNA 相互作用在病毒颗粒成熟中的作用，并证明了 CA 晶格在 保护靶细胞中的病毒核酸。 基于这些新颖的发现和广泛的初步数据，我们建议阐明其性质和 HIV-1 IN-gRNA 相互作用的规则，IN 与 gRNA 的结合如何介导 HIV-1 衣壳的正确组装 晶格以及受感染细胞如何感知和响应抑制 IN-gRNA 时产生的异常颗粒 CA 晶格的相互作用和不稳定。这些研究将填补我们理解的一个关键空白 HIV-1 IN 在颗粒成熟中的关键非催化功能以及抑制这些功能的后果 互动。总之，这个项目不仅将增强我们对 HIV-1 复制的基础知识，而且有助于 新型抗病毒药物的开发可以补充临床环境中基于 INSTI 的疗法。