The Influence of Host Factors on Retroviral Integration into Chromatin Templates

描述（由申请人提供）：逆转录病毒感染引起动物的多种癌症，以及人类的多种疾病，如白血病和获得性免疫缺陷综合征。整合过程对逆转录病毒的功能至关重要，但与整合过程相互作用并无疑影响整合过程的宿主因素仍然知之甚少。关于影响逆转录病毒在宿主基因组中选择整合位点的位置和方式的因素知之甚少，并且很少有整合酶相互作用蛋白被鉴定和验证。我们一直在研究逆转录病毒整合的长期目标是开发干预措施，可以调节的过程中对几个治疗目的，包括设计良性逆转录病毒为基础的基因靶向载体，避免插入诱变。鉴定出能促进整合到宿主基因组中的细胞因子，可能使我们能够设计整合反应的抑制剂，这一结果在设计抗HIV-1和其他抗逆转录病毒药物方面非常有用。本项目的目标是鉴定和表征与逆转录病毒整合酶相互作用并调节病毒整合结果的宿主因子。本研究使用基因治疗试验中最常用的逆转录病毒载体，莫洛尼鼠白血病病毒（MoMLV），作为体外和体内实验的模型逆转录病毒，我们在我们的研究计划中包括与HIV-1的比较研究。我们假设，宿主因素影响染色质结构的整合，通过与病毒整合酶蛋白直接相互作用的关键调制器。目标1：我们将确定转录诱导的染色质改变逆转录病毒整合模式的影响，使用我们的新的和创新的体内整合试验。在这个系统中，从广泛表征的小鼠乳腺肿瘤病毒长末端重复序列（MMTV LTR）的转录起始发生在响应类固醇激素，我们将使用这个功能来映射MoMLV整合事件与激素。目的2：我们还将确定整合酶相互作用的染色质相关宿主因子在整合和病毒感染性中的作用。我们已经确定了一些整合酶相互作用的宿主因子在cDNA屏幕上，我们将使用RNA干扰（RNAi）在哺乳动物细胞系敲除两种蛋白质，这两种蛋白质都是染色质结合因子。使用这种技术，我们表明，这些蛋白质之一抑制病毒的感染性。我们还将映射到嵌合牛乳头状瘤病毒-MMTV LTR附加体的整合事件在激素的存在和不存在下，在这些线。这些研究将通过确定至少两个关键的宿主因子和通过提供体内整合系统来推进逆转录病毒整合领域，该系统将使我们能够详细检查整合反应的具体步骤，并确定抗病毒治疗的新靶点。 公共卫生相关性：确定对整合至关重要的宿主因素很重要，原因有二。第一，设计良性的逆转录病毒基因打靶载体，以避免插入突变。其次，这些信息在设计抗艾滋病病毒、抗癌和其他抗逆转录病毒药物方面非常有用。

DESCRIPTION (provided by applicant): Retroviral infections cause a variety of cancers in animals, and a number of diverse diseases in humans such as leukemia and acquired immune deficiency syndrome. The process of integration is critical for retroviral function, yet the host factors that interface with, and undoubtedly influence the integration process are still poorly understood. Little is known about the factors influencing where and how retroviruses select integration sites within the genome of their hosts, and few integrase interacting proteins have been identified and verified. We have been studying retroviral integration with the long-term goal of developing interventions that can modulate the process towards several therapeutic ends, including design of benign retroviral-based gene targeting vectors and avoiding insertional mutagenesis. Identification of cellular factors that potentiate integration into host genomes may allow us to design inhibitors of the integration reaction, an outcome that would be extremely useful in designing anti-HIV-1 and other anti-retroviral drugs. The goal of this project is to identify and characterize host factors that interact with retroviral integrase and modulate the results of viral integration. This study uses the most commonly used retroviral vector in gene therapy trials, Moloney murine leukemia virus (MoMLV), as a model retrovirus for in vitro and in vivo experiments, and we include comparative studies with HIV-1 in our research plan. We hypothesize that host factors affecting chromatin structure are key modulators of integration via direct interactions with the viral integrase protein. Aim 1: We will determine the effect of transcription-induced chromatin alteration on retroviral integration patterns using our novel and innovative in vivo integration assay. In this system, transcription initiation from the extensively characterized mouse mammary tumor virus long terminal repeat (MMTV LTR) occurs in response to steroid hormones, and we will use this feature to map MoMLV integration events with and without hormone. Aim 2: We will also determine the role of integrase-interacting chromatin-associated host factors in integration and virus infectivity. We have identified a number of integrase-interacting host factors in cDNA screens, and we will use RNA interference (RNAi) in mammalian cell lines to knockdown two proteins initially, both of which are chromatin binding factors. Using this technique, we show that one of these proteins inhibits virus infectivity. We will also map integration events into the chimeric bovine papilloma virus-MMTV LTR episomes in the presence and absence of hormone in these lines. These studies will advance the retroviral integration field by identifying at least two key host factors and by providing an in vivo integration system that will enable us to examine specific steps of the integration reaction in detail, and identify new targets for antiviral therapy. PUBLIC HEALTH RELEVANCE: Identification of host factors critical for integration is important for two reasons. First, to design benign retroviral-based-gene targeting vectors to avoid insertional mutagenesis. Second, this information would be extremely useful in designing anti-HIV, anti-oncogenic, and other anti-retroviral drugs.