Mechanisms of retroviral integrase-DNA complexes assembly

逆转录病毒整合酶-DNA复合物组装机制

• 批准号: 10472029
• 负责人: Krishan Kumar Pandey
• 金额: $ 18.94万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-19 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10472029
• 关键词: Active Sites; Amino Acid Sequence; Architecture; Biological Models; C-terminal; Catalysis; Catalytic Domain; Complex; Core Assembly; Cryoelectron Microscopy; DNA; Data; Development; Dimerization; Distal; Genome; HIV Integrase; HIV-1; HIV-1 integrase; HIV-2; Image; Integrase; Integrase Inhibitors; Length; Lentivirus; Maps; Mediating; Molecular Conformation; N-terminal; Pathway interactions; Phenylalanine; Positioning Attribute; Property; Research; Resolution; Retroviridae; Role; Rous sarcoma virus; Route; SIV; Solubility; Structure; Synapses; System; Tail; Viral; Viral Genome; Virus Integration; Visna-maedi virus; alpha helix; dimer; electron density; flexibility; human pathogen; improved; inhibitor; insight; lentiviral integration; new therapeutic target; novel; particle; prevent; viral DNA

Abstract Retrovirus integrase (IN) is responsible for integration of viral genome into host DNA. IN multimerizes on viral DNA to produce intasomes. Intasomes produced from lentiviruses including HIV-1 demonstrate an extended architecture compared to other retroviral intasomes. The specific aims of this proposal are to: 1) determine the structure of HIV-2 intasomes by single-particle cryo-electron microscopy (cryo-EM) in the presence of HIV-1 IN strand transfer inhibitors (INSTIs); 2) determine the mechanisms of assembly of extended intasome architectures using HIV-1 and HIV-2 as model systems; and 3) produce and determine the structure of intasomes assembled with untagged native tetrameric HIV-1 and dimeric HIV-2 IN, as fusion tags used to enhance IN solubility may interfere with positioning of IN subunits in intasomes. As proof of concept and to demonstrate expertise, we determined the structure of Rous sarcoma virus octameric (8 IN subunits) cleaved synaptic complex intasome stabilized with INSTI MK-2048 at 3.21Å resolution by cryo-EM. We have obtained preliminary data for HIV-1 and HIV-2 intasomes structures using cryo-EM. The partial intasome structures from other research groups have further determined the mechanisms of HIV-1 INSTIs. However, the distal subunits which are crucial for intasome assembly and catalysis are not resolved in these structures. We will use wt HIV- 1 and HIV-2 IN with native N-terminal residue phenylalanine to produce intasomes for their complete structure determination by cryo-EM. HIV-2 is a significant human pathogen and determining of the structure of HIV-2 intasome with INTSIs will be complementary and confirmatory for HIV-1 studies. These studies will provide a greater understanding of intasome assembly mechanisms and facilitate development of active site and novel allosteric IN inhibitors.

抽象的 逆转录病毒整合酶（IN）负责将病毒基因组整合到宿主DNA中。 在病毒DNA上进行多种疾病以产生诱导。产生的诱导症 与其他 逆转录病毒。该提案的具体目的是：1）确定结构 在存在的情况下，单粒子冷冻电子显微镜（Cryo-EM）的HIV-2静态 链转移抑制剂中的HIV-1（Instis）； 2）确定组装机制 使用HIV-1和HIV-2作为模型系统的扩展Intasome架构； 3） 生产并确定与未标记天然组装的静态的结构 四聚体HIV-1和二聚体HIV-2 IN，因为用于增强溶解度的融合标签可能 干扰intomes中亚基的定位。 作为概念证明并证明了专业知识，我们确定了 ROUS肉瘤病毒八聚体（亚基8个）裂解突触复合体Intasome 通过Cryo-Em分辨率为3.21Å，用Insti MK-2048稳定。我们已经获得了 使用冷冻EM的HIV-1和HIV-2静态结构的初步数据。 来自其他研究小组的部分富含型结构已进一步确定 HIV-1学院的机制。但是，远端亚基至关重要 在这些结构中未能解决富含体组件和催化。我们将使用WT HIV- 1和HIV-2与天然N末端居住苯丙氨酸一起产生静态 它们通过冷冻EM确定的完整结构。 HIV-2是一种重要的人类病原体 并确定使用INTSI的HIV-2 Intasome的结构已完成 并确认HIV-1研究。这些研究将提供对 富含体组装机制，并促进活跃部位和新颖的发展 抑制剂变构。