Mechanisms of retroviral integrase-DNA complexes assembly

摘要 逆转录病毒整合酶(IN)负责病毒基因组与宿主DNA的整合。 在病毒DNA上进行多聚体以产生内切体。产生的整合体产自 与其他病毒相比，包括HIV-1在内的慢病毒表现出扩展的体系结构 逆转录病毒内切酶。这项建议的具体目的是：1)确定结构 用单粒子低温电子显微镜(Cryo-EM)检测HIV-2病毒内含体 链转移抑制物(INSTI)中HIV-1的作用；2)确定组装机制 使用HIV-1和HIV-2作为模型系统的扩展Intasome体系结构；和3) 产生并确定与未标记的原生基因组装的内聚体的结构 作为用于增强IN溶解性的融合标签的四聚体HIV-1和二聚体HIV-2 IN可 干扰肠小体中IN亚基的定位。 作为概念验证和展示专业知识，我们确定了 Rous肉瘤病毒八聚体(8IN亚基)裂解突触复合体 用INSTI MK-2048稳定在低温EM的3.21？分辨率下。我们已经获得了 利用低温电子显微镜获得HIV-1和HIV-2整合结构的初步数据。 来自其他研究小组的部分集合体结构进一步确定 HIV-1 INSTIs的机制。然而，远端亚单位对于 在这些结构中，内切酶的组装和催化作用并没有被分解。我们将使用wt HIV- 1和HIV-2与天然N-末端残基苯丙氨酸一起产生内切酶 用低温电子显微镜测定了它们的完整结构。HIV-2是一种重要的人类病原体 而用INTSI确定HIV-2连接体的结构将是互补的 对HIV-1研究具有确认性。这些研究将提供更多的了解 端粒体组装机制和促进活性部位的开发和新颖 变构蛋白抑制剂。

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.