The Influence of Host Factors on Retroviral Integration into Chromatin Templates

描述(申请人提供)：逆转录病毒感染可导致多种动物癌症，以及多种人类疾病，如白血病和获得性免疫缺陷综合症。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。人们对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，也很少识别和验证整合酶相互作用蛋白。我们一直在研究逆转录病毒的整合，长期目标是开发干预措施，以调节这一过程达到几个治疗目的，包括设计基于逆转录病毒的良性基因靶向载体，并避免插入突变。识别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，这一结果将在设计抗HIV-1和其他抗逆转录病毒药物方面非常有用。该项目的目标是确定和表征与逆转录病毒整合酶相互作用的宿主因子，并调节病毒整合的结果。本研究使用基因治疗实验中最常用的逆转录病毒载体Moloney小鼠白血病病毒(Moloney小鼠白血病病毒，MoMLV)作为体外和体内实验的逆转录病毒模型，并在我们的研究计划中包括与HIV-1的比较研究。我们假设，影响染色质结构的宿主因子是通过与病毒整合酶蛋白直接相互作用而整合的关键调节器。目的1：我们将使用我们新颖和创新的体内整合实验来确定转录诱导的染色质改变对逆转录病毒整合模式的影响。在这个系统中，被广泛描述的小鼠乳腺肿瘤病毒长末端重复序列(MMTV LTR)的转录起始发生在类固醇激素的响应下，我们将利用这一特征来定位MoMLV在激素作用下和不在激素作用下的整合事件。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。我们已经在cDNA筛选中鉴定了一些整合酶相互作用的宿主因子，并将在哺乳动物细胞系中使用RNA干扰(RNAi)最初敲除两个蛋白质，这两个蛋白质都是染色质结合因子。利用这项技术，我们证明了其中一种蛋白质可以抑制病毒的感染性。我们还将在这些株系中存在和不存在激素的情况下，将整合事件映射到嵌合的牛乳头瘤病毒-MMTV LTR episome中。这些研究将通过确定至少两个关键宿主因素和提供体内整合系统来推动逆转录病毒整合领域的发展，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：确定对融合至关重要的宿主因素很重要，原因有两个。首先，设计基于逆转录病毒的良性基因靶向载体，以避免插入突变。其次，这些信息在设计抗艾滋病毒、抗肿瘤和其他抗逆转录病毒药物方面将非常有用。

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.