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各种复制过程的动态结构。这个 HIV-1的成熟衣壳核心是由衣壳(CA)蛋白单体组成的晶格，被认为是 首先组装成CA二聚体，然后是~250个CA六聚体和12个CA五聚体。将CA组装成以下组件 表单需要每个CA单元的构象灵活性，从而产生独特的、可溶于溶剂的 与每个组件形式相关联的绑定表面。尽管我们对CA的理解取得了重大进展， 关于CA的结构动力学及其对病毒生物学的影响，还有许多悬而未决的问题。近期 工作支持在未成熟的六角体晶格的边缘存在部分六角体，这可能 作为蛋白质降解成熟的底物，并有助于GAG六角体晶格的组装。然而， 需要进一步的研究才能完全了解不成熟的Gag六角体晶格组装发生的顺序 以及它如何导致病毒蛋白水解酶的激活，以及重塑的组装机制 蛋白水解性地将CA裂解成成熟的衣壳核心。此外，成熟的衣壳核也可能经历 一定程度的重塑有助于逆转录、核进入和整合，尽管程度 所需解离的范围和解离开始的位置仍然存在争议。此外，我们仍然 不完全了解CA与宿主蛋白的广泛相互作用及其对病毒的影响 复制，以及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CA组装状态的分化，以评估它们对病毒复制事件的贡献。 适配子是与分子靶标结合的结构化寡核苷酸，可以选择用于区分 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 同样的蛋白质。我们已经确定了与HIV-1 CA六聚体晶格特异结合的适体，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。其中几个 这些适配子抑制HIV在细胞培养中的复制，表明它们与生物学上相关的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洞察 机制和相互作用，提出了新的病毒抑制策略，并确定了新的可访问的位置，以 告知治疗学的发展。在这里，我们建议确定关键的适体-CA相互作用在体外和 在体外和体内，研究抑制性适配子对艾滋病毒复制周期关键事件的影响， 并鉴定具有新的特异性和功能特性的适配子。这项工作将使这些应用成为可能 适体作为功能工具更好地了解HIV生物学和CA在HIV复制中的作用，识别 治疗靶点，并开发选择技术来识别具有所需功能的适配子 表型。