Aptamer tools for dissecting HIV-1 capsid function and identifying accessible, biologically relevant interaction surfaces.

用于剖析 HIV-1 衣壳功能并识别可访问的、生物学相关的相互作用表面的适体工具。

• 批准号: 10655852
• 负责人: Margaret J Lange
• 金额: $ 38.4万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-10 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10655852
• 关键词: Adopted; Adoption; Affinity; Amino Acids; Anti-HIV Agents; Antibodies; Binding; Binding Sites; Biological Assay; Biological Process; Biological Response Modifier Therapy; Biology; Biophysics; Biosensor; Capsid; Capsid Proteins; Cell Culture Techniques; Cell Nucleus; Cell physiology; Cells; Characteristics; Defect; Detection; Development; Discrimination; Dissociation; Drug Targeting; Enzymes; Equilibrium; Event; Foundations; Future; Genetic Materials; HIV; HIV-1; Immune response; In Vitro; Integration Host Factors; Link; Location; Mediating; Microscopy; Molecular; Molecular Conformation; Molecular Target; Mutagenesis; Mutation; Nuclear; Nuclear Import; Nucleic Acids; Oligonucleotides; Peptide Hydrolases; Phenotype; Play; Process; Property; Proteins; Public Health; RNA; Reagent; Replication-Associated Process; Research; Reverse Transcription; Role; Shapes; Site; Solvents; Specificity; Structure; Surface; System; Techniques; Therapeutic; Transcript; Viral; Viral Pathogenesis; Virus; Virus Activation; Virus Assembly; Virus Replication; Visualization; Work; aptamer; dimer; flexibility; in vivo; insight; monomer; novel; novel virus; programs; protein function; small molecule; therapeutic development; therapeutic target; three dimensional structure; tool; trafficking; virus genetics; virus host interaction

The HIV-1 capsid core is a dynamic structure that participates in a variety of replication processes. The mature viral capsid core of HIV-1 is a lattice composed of capsid (CA) protein monomers that are thought to assemble first into CA dimers, followed by ~250 CA hexamers and 12 CA pentamers. Assembly of CA into these forms requires conformational flexibility of each CA unit, resulting in the presence of unique, solvent-accessible binding surfaces associated with each assembly form. Despite significant advances in our understanding of CA, there are many unresolved questions regarding CA structural dynamics and their impact on viral biology. Recent work supports the presence of partial Gag hexamers at the edges of the immature hexamer lattice, which could serve as substrates for proteolytic maturation and contribute to assembly of the Gag hexamer lattice. However, further study is required to fully understand the order in which immature Gag hexamer lattice assembly occurs and how it leads to activation of the viral protease, as well as the assembly mechanisms underlying remodeling of proteolytically cleaved CA into the mature capsid core. In addition, the mature capsid core may also undergo some degree of remodeling to facilitate reverse transcription, nuclear entry and integration, although the degree of dissociation required and the location at which dissociation begins remain controversial. Furthermore, we still do not fully understand the broad spectrum of CA interactions with host proteins and their implications for virus replication, and previously undescribed interactions and targetable surfaces likely exist. Notably, limited tools exist for the differentiation of CA assembly states in vivo to assess their contributions to viral replication events. Aptamers are structured oligonucleotides that bind to molecular targets and can be selected to discriminate among very similar proteins, including those with only a single amino acid change or different conformations of the same protein. We have identified aptamers that specifically bind the HIV-1 CA hexamer lattice, as well as those that bind both the CA hexamer lattice and the soluble CA hexamer, but not the CA monomer. Several of these aptamers inhibit HIV replication in cell culture, suggesting that they bind to biologically relevant CA surfaces. Aptamer-mediated interrogation of these binding sites could provide insights into a variety of replication mechanisms and interactions, present new strategies for viral inhibition, and identify novel accessible sites to inform development of therapeutics. Here, we propose to identify key aptamer-CA interactions in vitro and in vivo, examine the impact of inhibitory aptamers on key events in the HIV replication cycle in vitro and in vivo, and to identify aptamers with new specificities and functional properties. This work will enable use of these aptamers as functional tools to better understand HIV biology and the role of CA in HIV replication, identify therapeutically targetable sites, and develop selection techniques to identify aptamers with desired functional phenotypes.

HIV-1衣壳核心是一种参与多种复制过程的动态结构。这 HIV-1 的成熟病毒衣壳核心是由衣壳 (CA) 蛋白单体组成的晶格，人们认为这些单体 首先组装成 CA 二聚体，然后是约 250 个 CA 六聚体和 12 个 CA 五聚体。将 CA 组装成这些 形式需要每个 CA 单元的构象灵活性，从而导致存在独特的、溶剂可访问的 与每个装配形式相关的结合表面。尽管我们对 CA 的理解取得了重大进展， 关于 CA 结构动力学及其对病毒生物学的影响还有许多未解决的问题。最近的 这项工作支持在未成熟的六聚体晶格边缘存在部分 Gag 六聚体，这可以 作为蛋白水解成熟的底物并有助于 Gag 六聚体晶格的组装。然而， 需要进一步研究以充分了解未成熟的 Gag 六聚体晶格组装发生的顺序 以及它如何导致病毒蛋白酶的激活，以及重塑背后的组装机制 蛋白水解裂解的 CA 进入成熟的衣壳核心。此外，成熟的衣壳核心也可能经历 一定程度的重塑以促进逆转录、核进入和整合，尽管程度 所需的解离程度和解离开始的位置仍然存在争议。此外，我们还 不完全了解 CA 与宿主蛋白相互作用的广谱及其对病毒的影响 复制、先前未描述的相互作用和可目标表面可能存在。值得注意的是，工具有限 存在用于区分体内 CA 组装状态，以评估它们对病毒复制事件的贡献。 适体是与分子靶标结合的结构化寡核苷酸，可以选择来区分 非常相似的蛋白质，包括那些只有一个氨基酸变化或不同构象的蛋白质 相同的蛋白质。我们已经鉴定出特异性结合 HIV-1 CA 六聚体晶格的适体，以及 那些既结合CA六聚体晶格又结合可溶性CA六聚体，但不结合CA单体的化合物。几个 这些适体抑制细胞培养中的 HIV 复制，表明它们与生物学相关的 CA 结合 表面。适体介导的对这些结合位点的询问可以提供对各种复制的见解 机制和相互作用，提出病毒抑制的新策略，并确定新的可访问位点 为治疗方法的开发提供信息。在这里，我们建议在体外和体内鉴定关键的适体-CA 相互作用 体内，检查抑制性适体对体外和体内 HIV 复制周期关键事件的影响， 并鉴定具有新特异性和功能特性的适体。这项工作将使这些 适体作为功能工具可以更好地了解 HIV 生物学和 CA 在 HIV 复制中的作用，识别 治疗靶向位点，并开发选择技术来识别具有所需功能的适体 表型。