Studies of the molecular mechanism of retroviral integration

逆转录病毒整合的分子机制研究

• 批准号: 8445867
• 负责人: Richard Fishel
• 金额: $ 19.14万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8445867
• 关键词: Affect; Anti-Retroviral Agents; Biochemical; Biological Assay; Biology; Chromatin; Chromatin Modeling; Chromosomes; Complementary DNA; Complex; DNA; DNA Binding; DNA Modification Process; Dissection; Drug Design; Drug Targeting; Engineering; Enzymes; Event; Foundations; Gammaretrovirus; Genetic Models; Genome; Goals; Grant; HIV; HIV Infections; HIV Integrase; Health; Hela Cells; Heterogeneity; Histone H2A; Histones; Human T-Cell Leukemia Viruses; In Vitro; Infection; Integrase; Kinetics; Length; Life Cycle Stages; Mechanics; Mediating; Modeling; Molecular; Mutation; Nucleosomes; Pharmaceutical Preparations; Pharmacotherapy; Post-Translational Protein Processing; Process; Proteins; Reaction; Recombinants; Reporting; Resistance; Resistance development; Retroviridae; Site; Solid; Spumavirus; Structural Models; Structure; Subfamily lentivirinae; System; Systems Analysis; Tail; Therapeutic; Time; Vertebral column; Viral; Viral Proteins; Virus Integration; Visual; chromatin modification; clinically relevant; comparative; dimer; flexibility; histone modification; in vivo; inhibitor/antagonist; innovation; mutant; novel; pandemic disease; prototype; public health relevance; reconstitution; resistance mutation; single molecule; time use; viral DNA

DESCRIPTION (provided by applicant): Retroviral infections, including HIV and HTLV, continue to be a pandemic problem. While several drug therapies are able to treat HIV infection, the viral propensity to develop resistance mutations remains challenging. Novel drug targets remain a priority for the HIV treatment arsenal. The most recent class of anti-retroviral drugs target the HIV enzyme integrase. These drugs also inhibit the prototype foamy virus (PFV) integrase. Stable integration of the viral cDNA into the host chromosome is required for a productive retroviral infection and is mediated by an integration complex. The mechanics of monomeric retroviral integrase proteins with model viral and target DNA do not recapitulate the integration reaction of a tetrameric integration complex. Importantly, PFV integrase readily forms physiologically relevant tetramers with model viral DNAs in vitro and is the only integrase where the crystal structure has been solved with viral and target DNAs. We propose to analyze the kinetic and biophysical mechanism of retroviral integration with recombinant PFV integrase as a structural and genetic model. Several innovative single molecule analytical systems will be used to visualize and differentiate the two independent strand transfer events in real time. In addition we will examine for the first time nucleosome DNA targets containing histones with specific post-translational modifications that model chromatin DNA targets in vivo. This proposal contains two highly focused Specific Aims: 1.) Real time analysis of retroviral integration and 2.) Analysis of DNA and chromatin modifications on integration. We will first detail real-time PFV integration into single DNA molecules. Once we have established the kinetics and mechanism of PFV integration into naked DNA, we will introduce increasingly complex physiologically relevant components that include defined poly- nucleosome DNA as well as DNA targets with increased flexibility. Ultimately, this exploratory grant will provide a quantitative and visual platform for probing the detailed kinetics and mechanism of pathogenic retroviral integration, such as HIV.

描述（由申请人提供）：逆转录病毒感染，包括 HIV 和 HTLV，仍然是一个流行病。虽然几种药物疗法能够治疗艾滋病毒感染，但病毒产生耐药突变的倾向仍然具有挑战性。新的药物靶点仍然是艾滋病毒治疗的优先事项。最新一类抗逆转录病毒药物以 HIV 酶整合酶为靶点。这些药物还抑制原型泡沫病毒（PFV）整合酶。病毒 cDNA 稳定整合到宿主染色体中是有效逆转录病毒感染所必需的，并且由整合复合物介导。单体逆转录病毒整合酶蛋白与模型病毒和靶 DNA 的机制并不能概括四聚体整合复合物的整合反应。重要的是，PFV 整合酶很容易在体外与模型病毒 DNA 形成生理相关的四聚体，并且是唯一一种用病毒和靶 DNA 解析晶体结构的整合酶。我们建议使用重组 PFV 整合酶作为结构和遗传模型来分析逆转录病毒整合的动力学和生物物理机制。几种创新的单分子分析系统将用于实时可视化和区分两个独立的链转移事件。此外，我们将首次检查含有具有特定翻译后修饰的组蛋白的核小体 DNA 靶标，这些组蛋白可模拟体内染色质 DNA 靶标。该提案包含两个高度集中的具体目标：1.) 逆转录病毒整合的实时分析和 2.) 分析 DNA 和染色质修饰对整合的影响。我们将首先详细介绍实时 PFV 整合到单个 DNA 分子中的情况。一旦我们确定了 PFV 整合到裸露 DNA 中的动力学和机制，我们将引入日益复杂的生理相关成分，其中包括确定的多核小体 DNA 以及具有更高灵活性的 DNA 靶标。最终，这笔探索性资助将为探索致病性逆转录病毒整合（例如艾滋病毒）的详细动力学和机制提供一个定量和可视化平台。