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衣壳核心是一种动态结构，参与多种复制过程。的 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， 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，导致存在独特的，溶剂可接近的 绑定与每个组件表单相关联的表面。尽管我们对CA的理解有了很大的进步， 关于CA结构动力学及其对病毒生物学的影响，存在许多未解决的问题。最近 工作支持在未成熟六聚体晶格的边缘存在部分Gag六聚体，这可能 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，在这方面， 需要进一步的研究来完全理解未成熟的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，虽然程度 解离所需的时间和解离开始的位置仍然存在争议。此外，我们还 尚不完全了解CA与宿主蛋白质的广泛相互作用及其对病毒的影响 复制，以及以前未描述的相互作用和目标表面可能存在。值得注意的是，工具有限 存在的CA组装状态在体内的分化，以评估其对病毒复制事件的贡献。 适体是结构化的寡核苷酸，其结合分子靶标并且可以被选择以区分 在非常相似的蛋白质中，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。我们已经鉴定了特异性结合HIV-1 CA六聚体晶格的适体，以及 结合CA六聚体晶格和可溶性CA六聚体但不结合CA单体的那些。几 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，提出了新的策略，抑制病毒，并确定新的可访问的网站， 为治疗学的发展提供信息。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复制周期中关键事件的影响， 并鉴定具有新特异性和功能特性的适体。这项工作将使使用这些 适体作为功能工具，以更好地了解艾滋病毒生物学和CA在艾滋病毒复制中的作用， 治疗靶向位点，并开发选择技术以鉴定具有所需功能的适体。 表型