BIOCHEMICAL MECHANISM OF HIV DNA INTEGRATION

HIV DNA 整合的生化机制

• 批准号: 6373509
• 负责人: Alan N. Engelman
• 金额: $ 34.19万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6373509
• 关键词: DNA binding protein; DNA footprinting; genetic promoter element; human immunodeficiency virus 1; integrase; polymerase chain reaction; protein purification; protein structure function; site directed mutagenesis; virus DNA; virus infection mechanism

Human immunodeficiency virus type 1 (HIV-1) must integrate the cDNA copy of its RNA genome into an infected cell chromosome in order to efficiently replicate. The key viral players in integration are the trans-acting integrase (IN) protein, which enters the cell as a part of the virus, and the cis-acting DNA attachment (att) site, comprised of the ends of linear cDNA made by reverse transcription. In cells, integration is mediated through large subviral nucleoprotein preintegration complexes (PICs) that are derived from the cores of infecting virions. PICs isolated from infected cells can integrate their endogenous cDNA into an added target DNA in vitro. PICs were previously purified based on their large size. Sizing on its own is not a very efficient purification technique: it is inherently diluting, and large cellular assemblies copurify with PICs. Novel techniques based on affinity chromatography will be developed to increase the purity and yield of PICs approximately 100-fold over existing techniques. In addition to IN, host factors play essential roles in the structure and function of retroviral PICs. Two host proteins, HMG I(Y) and the barrier-to-autointegration factor (BAF), were previously implicated in HIV-1 integration, but it unclear which of these is physiologically-relevant. The stoichiometries of HMG I(Y) and BAF to IN and cDNA will be determined in purified PICs. Other viral proteins, including matrix, reverse transcriptase, Vpr, and nucleocapsid cofractionate with HIV-1 PICs. Although not needed for DNA recombination, matrix and Vpr may facilitate the nuclear import of PICs in nondividing cells. The stoichiometries of these viral proteins will be determined in purified samples, along with the folding topologies of IN, matrix and reverse transcriptase. Current models of protein-protein interactions important for PIC structure and function are not well defined, but predict the two cDNA ends are coupled for IN catalysis in vivo. Preliminary data supports a model where the cDNA ends are uncoupled for an initial catalytic step, and then come together through protein-protein interactions for integration. The contribution of viral and host factors to these interactions will be determined. A novel IN mutant was identified that in addition to normal integration promotes the integration of just one cDNA end at a time, a pathway that is deleterious to virus growth. These mutant PICs will be analyzed in detail to identify protein-protein and protein-DNA interactions required for normal PIC function. The results of these experiments will be used to formulate a detailed model of the structural and functional organization of HIV-1 PICs, which will aid the design of antiviral drugs targeted against HIV-1 integration.

1 型人类免疫缺陷病毒 (HIV-1) 必须将其 RNA 基因组的 cDNA 拷贝整合到受感染的细胞染色体中才能有效复制。 病毒整合中的关键角色是反式作用整合酶 (IN) 蛋白（它作为病毒的一部分进入细胞）和顺式作用 DNA 附着 (att) 位点（由逆转录产生的线性 cDNA 末端组成）。 在细胞中，整合是通过源自感染病毒粒子核心的大型亚病毒核蛋白预整合复合物（PIC）介导的。从受感染细胞中分离的 PIC 可以在体外将其内源 cDNA 整合到添加的靶 DNA 中。由于 PIC 尺寸较大，因此之前对其进行了纯化。 单独确定尺寸并不是一种非常有效的纯化技术：它本质上是稀释的，并且大型细胞组装体会与 PIC 共纯化。 将开发基于亲和色谱的新技术，以将 PIC 的纯度和产量提高到现有技术的约 100 倍。除 IN 外，宿主因子在逆转录病毒 PIC 的结构和功能中也发挥着重要作用。 两种宿主蛋白​​ HMG I(Y) 和自动整合屏障因子 (BAF) 先前被认为与 HIV-1 整合有关，但尚不清楚其中哪一个具有生理相关性。 HMG I(Y) 和 BAF 与 IN 和 cDNA 的化学计量将在纯化的 PIC 中确定。其他病毒蛋白，包括基质、逆转录酶、Vpr 和核衣壳，与 HIV-1 PIC 共分级。 虽然 DNA 重组不需要，但基质和 Vpr 可以促进 PIC 在非分裂细胞中的核输入。 这些病毒蛋白的化学计量以及 IN、基质和逆转录酶的折叠拓扑将在纯化样品中确定。目前对 PIC 结构和功能很重要的蛋白质-蛋白质相互作用模型尚未明确定义，但预测两个 cDNA 末端在体内偶联以进行 IN 催化。 初步数据支持这样一种模型，其中 cDNA 末端在初始催化步骤中解偶联，然后通过蛋白质-蛋白质相互作用聚集在一起进行整合。 病毒和宿主因素对这些相互作用的贡献将被确定。 鉴定出一种新的 IN 突变体，除了正常整合之外，一次仅促进一个 cDNA 末端的整合，这是一种对病毒生长有害的途径。 将详细分析这些突变 PIC，以确定正常 PIC 功能所需的蛋白质-蛋白质和蛋白质-DNA 相互作用。 这些实验的结果将用于制定 HIV-1 PIC 的结构和功能组织的详细模型，这将有助于设计针对 HIV-1 整合的抗病毒药物。