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.

摘要 逆转录病毒整合酶（IN）负责将病毒基因组整合到宿主DNA中。 IN在病毒DNA上多聚化以产生整合体。从以下物质生产的整合体 包括HIV-1在内的慢病毒与其他慢病毒相比， 逆转录病毒整合体。本建议的具体目标是：1）确定结构 通过单粒子冷冻电子显微镜（cryo-EM）在存在 HIV-1 IN链转移抑制剂（INSTIs）; 2）确定组装机制 使用HIV-1和HIV-2作为模型系统的扩展整合体结构;以及3） 产生并确定与未标记的天然蛋白质组装的整合体的结构 四聚体HIV-1和二聚体HIV-2 IN，作为用于增强IN溶解度的融合标签， 干扰IN亚基在整合体中的定位。 作为概念验证和专业知识展示，我们确定了 劳斯肉瘤病毒八聚体（8IN亚单位）裂解的突触复合体 通过cryo-EM以3.21 μ m的分辨率用MIBI MK-2048稳定。我们所获得 使用冷冻-EM的HIV-1和HIV-2整合体结构的初步数据。 其他研究小组的部分整合体结构进一步确定了 HIV-1 INSTI的机制。然而，对于细胞生长至关重要的远端亚基 在这些结构中不能解决整合体组装和催化。我们将使用野生型艾滋病毒- 1和HIV-2 IN与天然N-末端残基苯丙氨酸，以产生用于 用冷冻电镜测定其完整结构。HIV-2是一种重要的人类病原体 用INTSIs测定HIV-2整合体的结构将是一种补充 和HIV-1研究的确证性。这些研究将使人们更好地了解 整合体组装机制并促进活性位点和新 变构IN抑制剂。