Integration of Retroviral DNA--Accessing Host Target DNA

描述（由申请人提供）：本提案的长期目标是 详细了解逆转录病毒DNA如何获得其 在宿主染色体中的整合靶点，以及整合反应是如何 在细胞中完成。禽肉瘤/白血病病毒（ASV）是主要的 这些研究的模型。然而，使用可以整合的ASV衍生物， 它的DNA进入小鼠和人类，允许-获得广泛的遗传基础， 可用于哺乳动物系统的生化信息，以及 与鼠（MLV）和人（HIV-1）逆转录病毒的比较。两个互补 将追求具体目标。在特异性目标1中，促进 ASV整合前复合体的核进入及其与宿主的关联 染色质将被鉴定和表征：（a）监测染色质的系统。 细胞周期对逆转录病毒复制和主动核输入的影响 将开发和使用病毒DNA的关键特征， ASV与MLV和HIV- 1，-研究ASV整合酶的作用 (IN)核定位信号（NLS）介导病毒进入核 DNA. (b)与NLS中的ASV相互作用以促进 核进入将被识别和表征，（c）假设， ASV IN相互作用细胞蛋白（Daxx）在整合中起作用， 测试.具体目标2将研究宿主细胞功能在以下方面的作用： 逆转录病毒DNA整合，并基于最近的遗传证据， 一个实验室涉及细胞，非同源末端连接（NHEJ）DNA 逆转录病毒DNA整合中的修复途径（丹尼尔等人，科学284：644， 1999年）：（a）假设-病毒DNA的3 '-末端与宿主DNA的连接 是引发NHEJ反应所必需的，并且需要NHEJ的组成部分 对于随后将病毒的5 '端连接到宿主DNA，将 （B）逆转录病毒与病毒载体之间的物理和功能相互作用 将分析NHEJ的组成部分，（c）早期事件如何发生的问题 导致逆转录病毒DNA整合可能会影响其他蛋白质， 使用蛋白质组学来解决。一系列最先进的基因 将采用生物化学和细胞生物学方法。逆转录病毒DNA 整合是逆转录病毒复制周期中的重要步骤， 也对它们的致病性有很大贡献。重点是 病毒-宿主细胞相互作用，这些研究将揭示分子机制 与逆转录病毒和细胞生物学有关，包括核输入和 对基因组稳定性至关重要的细胞功能。这些研究还可能 提出了预防或治疗逆转录病毒疾病的新策略， 细胞和病毒的功能。

DESCRIPTION (provided by applicant): The long term goals of this proposal are to develop a detailed understanding of how retroviral DNA gains access to its integration targets in the host chromosome, and how the integration reaction is completed in the cell. The avian sarcoma/leukosis virus (ASV) is the primary model for these studies. However, use of an ASV derivative that can integrate its DNA into mouse and human, allows - access to the broad base of genetic and biochemical information available for mammalian systems, and - direct comparisons with murine (MLV) and human (HIV-1) retroviruses. Two complementary Specific Aims will be pursued. In Specific Aim 1 host proteins that facilitate nuclear entry of the ASV pre-integration complex and its association with host chromatin will be identified and characterized: (a) Systems to monitor the effects of the cell cycle on retroviral replication and active nuclear import of viral DNA will be developed and used - to compare the critical features of ASV with MLV and HIV- 1 and, - to investigate the role of the ASV integrase (IN) nuclear localization signal (NLS) in mediating the nuclear entry of viral DNA. (b) Cellular proteins that interact with the ASV IN NLS to facilitate nuclear entry will be identified and characterized and, (c) The hypothesis that an ASV IN-interacting cellular protein (Daxx) has a role in integration will be tested. Specific Aim 2 will investigate the role of host cell functions in retroviral DNA integration and is based on recent genetic evidence from this laboratory implicating the cellular, non-homologous end-joining (NHEJ) DNA repair pathway in retroviral DNA integration (Daniel eta!., Science 284: 644, 1999): (a) The hypotheses that - joining of the 3'-end of viral DNA to host DNA is required to elicit an NHEJ response and - components of NHEJ are required for subsequent joining of the 5'-ends of viral to host DNA, will be investigated; (b) Physical and functional interactions between retroviral and NHEJ components will be analyzed and, (c) The question of how early events leading to retroviral DNA integration may affect other proteins will be addressed using proteomics. A wide range of state-of-the-art genetic, biochemical, and cell biological methodologies will be employed. Retroviral DNA integration is an essential step in the replication cycle of retroviruses; it also contributes significantly to their pathogenicity. With a focus on virus-host cell interactions, these studies will reveal molecular mechanisms relevant to both retroviral and cellular biology, including nuclear import and cellular functions that are critical for genome stability. The studies may also suggest new strategies to prevent or treat retroviral disease by targeting cellular as well as viral functions.