HIV integrase/DNA complexes and concerted integration

HIV整合酶/DNA复合物和协同整合

• 批准号: 7622272
• 负责人: DUANE P GRANDGENETT
• 金额: $ 20.28万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-05 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7622272
• 关键词: Abbreviations; Address; Affect; Amino Acids; Atomic Force Microscopy; Binding; Biochemical; Biological Models; Cell Nucleus; Cells; Chemicals; Chromosomes; Circular DNA; Cleaved cell; Complementary DNA; Complex; Computer Simulation; Consensus Sequence; Coupled; Cytoplasm; DNA; DNA Integration; DNA Sequence; Defect; Digestion; Dinucleoside Phosphates; Dipeptides; Docking; Fluorescence Resonance Energy Transfer; Fostering; Gel; Genome; HIV-1; In Vitro; Integrase; Investigation; Knowledge; Label; Long Terminal Repeats; Macromolecular Complexes; Mass Spectrum Analysis; Measurement; Measures; Modeling; Molecular; Molecular Structure; Nuclear; Nucleoproteins; Pathway interactions; Pattern; Process; Property; Proteins; Proviruses; Sepharose; Site; Site-Directed Mutagenesis; Structure; Structure-Activity Relationship; Superhelical DNA; Synapses; Techniques; Terminal Repeat Sequences; Testing; Time; Viral; Viral Proteins; Virus; crosslink; dimer; fluorophore; in vivo; insight; mutant; nucleocytoplasmic transport; public health relevance; reconstitution; research study; transcriptional coactivator p75; viral DNA; viral RNA

DESCRIPTION (provided by applicant): In HIV-1 infected cells, the viral RNA is reverse transcribed into a 10 kbp linear blunt-ended DNA followed by the formation of the cytoplasmic preintegration complex (PIC). The viral integrase (IN) within the PIC cleaves a dinucleotide from the 3' OH ends of the viral DNA prior to nuclear transport. IN inserts the two recessed ends in a concerted fashion into the host chromosome, producing the provirus. The cellular co- factor, LEDGF/p75, influences the ability of the PIC to select sites on the host chromosomes for integration. The IN-IN and IN-DNA interactions within the PIC critical for concerted integration and the effect of cellular co-factors on this process are not well defined at the biochemical and biophysical levels. We recently identified a nucleoprotein complex on native agarose gels where IN non-covalently juxtaposes two viral DNA ends that produces the synaptic complex (SC) that possesses properties associated with the PIC. We will study the assembly properties of the SC, investigate the ability of IN to protect the terminal U5 and U3 DNA sequences from DNaseI digestion and determine the molar ratios of chemically cross-linked dimers, tetramers and a larger-size multimer of IN located in the SC. A unique ~32 bp DNaseI protective footprint by IN in the SC suggests a structural relationship to the larger-size multimer. We will investigate the relationships between these cross-linked species to identify the unique inter-subunit residues responsible for formation of the tetramer, as determined by mass spectrometry studies of cross-linked tryptic dipeptides. In- gel fluorescence resonance energy transfer-derived distance measurements and atomic force microscopy will determine the topology of the two viral DNA ends within the SC. We expect to generate a structural paradigm describing the SC and its relationship to the PIC. We will define the cellular co-factor LEDGF/p75 physical interactions with the SC and determine if it affects the SC by measuring the distance between fluorophore- labeled DNA substrates in the SC. We will develop a model system to determine if LEDGF/p75 influences host-site selection on chromatinized supercoiled target DNA for concerted integration. We will study known Class II IN mutants in vitro that are catalytically active and possess post-nuclear entry defects. We will also determine the functionality of the unique residues identified in the cross-linked IN tetramer observed in the SC. Site-directed mutagenesis of these IN residues will be performed for probing of the PIC in vivo and the SC in vitro. The knowledge gain from the R21 studies will be used to further investigate the PIC and the involvement of cellular co-factors in integration. Completion of the R21/R33 studies will foster a necessary and achievable understanding of concerted integration both in vitro and in vivo. PUBLIC HEALTH RELEVANCE: We are building a platform of structural information to understand the HIV-1 synaptic complex (SC) capable of concerted integration. We will determine the IN subunit interactions within SC to define what residues are responsible for formation of the IN tetramer. The functionality of these residues will be investigated by site-directed mutagenesis to probe the PIC in vivo and the SC in vitro. We will determine if LEDGF/p75 physically interacts with SC and test whether it influences host-site selection on chromatinized supercoiled target DNA that contains HIV-1 host-site consensus sequences.

描述（由申请人提供）：在HIV-1感染的细胞中，病毒RNA被逆转录成10kbp线性平端DNA，随后形成细胞质预整合复合物（PIC）。在核转运之前，PIC 内的病毒整合酶 (IN) 从病毒 DNA 的 3' OH 末端裂解一个二核苷酸。 IN以一致的方式将两个凹进的末端插入宿主染色体，产生原病毒。细胞辅因子 LEDGF/p75 影响 PIC 选择宿主染色体上进行整合的位点的能力。 PIC 内的 IN-IN 和 IN-DNA 相互作用对于协同整合至关重要，并且细胞辅助因子对此过程的影响在生化和生物物理水平上尚未得到很好的定义。我们最近在天然琼脂糖凝胶上发现了一种核蛋白复合物，其中 IN 非共价并置两个病毒 DNA 末端，产生具有与 PIC 相关特性的突触复合物 (SC)。我们将研究 SC 的组装特性，研究 IN 保护末端 U5 和 U3 DNA 序列免遭 DNaseI 消化的能力，并确定位于 SC 中的化学交联二聚体、四聚体和较大尺寸的 IN 多聚体的摩尔比。 SC 中 IN 独特的约 32 bp DNaseI 保护足迹表明与较大尺寸的多聚体存在结构关系。我们将研究这些交联物种之间的关系，以确定负责形成四聚体的独特亚基间残基，这是通过交联胰蛋白酶二肽的质谱研究确定的。凝胶内荧光共振能量转移衍生的距离测量和原子力显微镜将确定 SC 内两个病毒 DNA 末端的拓扑结构。我们期望生成一个描述 SC 及其与 PIC 关系的结构范式。我们将定义细胞辅因子 LEDGF/p75 与 SC 的物理相互作用，并通过测量 SC 中荧光团标记的 DNA 底物之间的距离来确定它是否影响 SC。我们将开发一个模型系统来确定 LEDGF/p75 是否影响染色质超螺旋靶 DNA 的宿主位点选择以实现协调整合。我们将在体外研究已知的 II 类 IN 突变体，这些突变体具有催化活性并具有入核后缺陷。我们还将确定在 SC 中观察到的交联 IN 四聚体中识别出的独特残基的功能。这些 IN 残基的定点诱变将用于体内 PIC 和体外 SC 的探测。从 R21 研究中获得的知识将用于进一步研究 PIC 以及细胞辅助因子在整合中的参与。 R21/R33 研究的完成将促进对体外和体内协同整合的必要且可实现的理解。公共健康相关性：我们正在构建一个结构信息平台，以了解能够协调整合的 HIV-1 突触复合体 (SC)。我们将确定 SC 内的 IN 亚基相互作用，以确定哪些残基负责形成 IN 四聚体。这些残基的功能将通过定点诱变进行研究，以探测体内的 PIC 和体外的 SC。我们将确定 LEDGF/p75 是否与 SC 发生物理相互作用，并测试它是否影响包含 HIV-1 宿主位点共有序列的染色质超螺旋靶 DNA 的宿主位点选择。