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 CAPSID核心是一种动态结构，参与各种复制过程。这 HIV-1的成熟病毒capsid核心是一个由衣壳（CA）蛋白质单体组成的晶格，被认为是 首先组装成Ca二聚体，然后将〜250 Ca六聚体和12个Ca pentamers组装。将CA组装成这些 表格需要每个CA单元的构象柔韧性，导致存在独特的，溶剂可访问 与每个组件形式关联的绑定表面。尽管我们对CA的理解取得了重大进展，但 关于CA结构动态及其对病毒生物学的影响，有许多未解决的问题。最近的 工作支持未成熟六聚体晶格边缘的部分堵塞六聚体，这可以 用作蛋白水解成熟的底物，并有助于hexamer晶格的组装。然而， 需要进一步的研究以充分了解未成熟hexamer晶格组件的顺序 以及它如何导致病毒蛋白酶的激活以及重塑基础机制 蛋白水解裂解的Ca进入成熟的衣壳芯。此外，成熟的衣壳芯也可能发生 一定程度的重塑以促进逆转录，核进入和整合 所需的解离和解离的位置仍存在争议。此外，我们仍然 不要完全了解CA与宿主蛋白的广泛相互作用及其对病毒的影响 复制，以前未描述的相互作用和可靶向表面可能存在。值得注意的是，工具有限 存在于体内的CA组装状态以评估其对病毒复制事件的贡献的存在。 适体是结合与分子靶标结合的结构化寡核苷酸，可以选择以区分 在非常相似的蛋白质中，包括只有单个氨基酸变化或不同构象的蛋白质 相同的蛋白质。我们已经确定了特异性结合HIV-1 CA六聚体晶格的适体 那些绑定Ca六聚体晶格和可溶性CA六聚体的人，而不是Ca单体。几个 这些适体在细胞培养中抑制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审问可以为各种复制提供见解 机制和相互作用，提出病毒抑制的新策略，并确定新颖的可访问地点 告知治疗学的开发。在这里，我们建议在体外和在 体内，检查抑制性适体对艾滋病毒复制周期中关键事件的影响，体外和体内， 并确定具有新的特异性和功能特性的适体。这项工作将有助于使用这些 适体作为功能工具，以更好地了解HIV生物学和CA在HIV复制中的作用，确定 具有治疗目标的站点，并开发选择技术以识别具有所需功能的适体 表型。