BIOCHEMICAL MECHANISM OF HIV DNA INTEGRATION

HIV DNA 整合的生化机制

• 批准号: 6631817
• 负责人: Alan N. Engelman
• 金额: $ 34.06万
• 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/6631817
• 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.

人类免疫缺陷病毒1型（HIV-1）必须将其RNA基因组的cDNA拷贝整合到受感染细胞的染色体中才能有效地复制。整合中的关键病毒参与者是反式作用整合酶（in）蛋白，它作为病毒的一部分进入细胞，以及顺式作用DNA附着位点（att），由逆转录产生的线性cDNA末端组成。在细胞中，整合是通过来自感染病毒粒子核心的大型亚病毒核蛋白预整合复合物（PICs）介导的。从感染细胞中分离的PICs可以在体外将其内源性cDNA整合到添加的靶DNA中。PICs以前是基于它们的大尺寸纯化的。施胶本身并不是一种非常有效的净化技术：它本质上是稀释的，而且大的细胞组件与PICs共混。将开发基于亲和层析的新技术，使PICs的纯度和收率比现有技术提高约100倍。除了In外，宿主因子在逆转录病毒PICs的结构和功能中也起着重要作用。两种宿主蛋白，HMG I(Y)和自整合障碍因子（BAF），先前与HIV-1整合有关，但尚不清楚其中哪一种与生理相关。在纯化的PICs中测定HMG I(Y)和BAF对IN和cDNA的化学计量学。其他病毒蛋白，包括基质、逆转录酶、Vpr和核衣壳与HIV-1 PICs发生分离。虽然不需要DNA重组，但基质和Vpr可能促进非分裂细胞中PICs的核输入。这些病毒蛋白的化学计量学将在纯化样品中测定，同时测定in、基质和逆转录酶的折叠拓扑结构。目前对PIC结构和功能重要的蛋白质-蛋白质相互作用的模型尚未很好地定义，但预测两个cDNA末端在体内偶联以催化IN。初步数据支持一种模型，即cDNA末端在最初的催化步骤中解耦，然后通过蛋白质-蛋白质相互作用整合在一起。病毒和宿主因素对这些相互作用的贡献将被确定。发现了一种新的IN突变体，除了正常整合外，它一次只促进一个cDNA末端的整合，这是一种对病毒生长有害的途径。这些突变的PIC将被详细分析，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。这些实验结果将用于制定HIV-1 PICs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。