The Influence of Host Factors on Retroviral Integration into Chromatin Templates

描述（由申请人提供）：逆转录病毒感染在动物中引起各种癌症，在人类中引起许多不同的疾病，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，然而与整合过程交互并无疑影响整合过程的宿主因子仍然知之甚少。对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，其长期目标是开发干预措施，以调节这一过程，实现多种治疗目的，包括设计基于逆转录病毒的良性基因靶向载体和避免插入突变。鉴定能够增强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，这一结果将对设计抗hiv -1和其他抗逆转录病毒药物非常有用。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Moloney小鼠白血病病毒（MoMLV）作为模型逆转录病毒进行体外和体内实验，并在研究计划中纳入与HIV-1的比较研究。我们假设影响染色质结构的宿主因子通过与病毒整合酶蛋白的直接相互作用是整合的关键调节剂。目的1：我们将利用我们新颖创新的体内整合试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，广泛表征的小鼠乳腺肿瘤病毒长末端重复序列（MMTV LTR）的转录起始是在类固醇激素的作用下发生的，我们将利用这一特征来绘制有激素和没有激素的MoMLV整合事件。目的2：我们还将确定整合酶相互作用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筛选中发现了许多整合酶相互作用的宿主因子，我们将在哺乳动物细胞系中使用RNA干扰（RNAi）来初始敲除两种蛋白质，这两种蛋白质都是染色质结合因子。利用这种技术，我们发现其中一种蛋白质抑制了病毒的传染性。我们还将在这些品系中存在和不存在激素的嵌合牛乳头瘤病毒- mmtv LTR发作中绘制整合事件。这些研究将通过确定至少两个关键的宿主因子和提供一个体内整合系统来推进逆转录病毒整合领域，这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。

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.