The Influence of Host Factors on Retroviral Integration into Chromatin Templates

宿主因素对逆转录病毒整合到染色质模板中的影响

基本信息

批准号：
8085731
负责人：
Barbara Studamire
金额：
$ 13.85万
依托单位：
BROOKLYN COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-15 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8085731
关键词：
Acquired Immunodeficiency Syndrome Address Affect Animals Anti-Retroviral Agents Antiviral Therapy Applications Grants Benign Binding Binding Proteins Biological Assay Biology Bovine Papillomavirus Bromodomain Containing Protein 2 Cell Line Cells Chairperson Chromatin Chromatin Structure Collaborations Comparative Study Complementary DNA Data Development Disease Enhancers Environment Episome Event Exhibits Funding Future Gene Targeting Genes Genetic Transcription Genome Goals HIV HIV-1 Homologous Gene Hormone Responsive Hormones Human In Vitro Infection Insertional Mutagenesis Integrase Integration Host Factors Intervention Knowledge Laboratories Long Terminal Repeats Malignant Neoplasms Mammalian Cell Maps Mentors Methodology Modeling Molecular Conformation Moloney Leukemia Virus Mouse Mammary Tumor Virus Mus Oncogenic Outcome Pattern Pharmaceutical Preparations Phenotype Principal Investigator Process Proteins Proviruses Publications RNA Interference Reaction Research Research Personnel Resources Retroviral Vector Retroviridae Role Science Scientist Site Structure Students System Systems Integration Techniques Terminal Repeat Sequences Testing Therapeutic Time Training Transcription Initiation Transcriptional Activation Universities Viral Virus Virus Diseases Virus Integration Yeasts base career chromatin remodeling college design gene therapy genome-wide in vivo inhibitor/antagonist innovation interest knock-down leukemia meetings member novel peer preference prevent professor public health relevance research study response skills steroid hormone therapeutic gene vector yeast two hybrid system

项目摘要

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.

描述（由申请人提供）：逆转录病毒感染会导致动物的多种癌症以及人类的多种疾病，例如白血病和获得性免疫缺陷综合症。整合过程对于逆转录病毒功能至关重要，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。对于影响逆转录病毒在宿主基因组内的位置和方式选择整合位点的因素知之甚少，并且很少有整合酶相互作用蛋白被识别和验证。我们一直在研究逆转录病毒整合，其长期目标是开发可以调节这一过程以实现多种治疗目的的干预措施，包括设计良性基于逆转录病毒的基因靶向载体和避免插入突变。鉴定出增强与宿主基因组整合的细胞因子可能使我们能够设计整合反应的抑制剂，这一结果对于设计抗 HIV-1 和其他抗逆转录病毒药物非常有用。该项目的目标是识别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，并且我们将与HIV-1的比较研究纳入我们的研究计划中。我们假设影响染色质结构的宿主因素是通过与病毒整合酶蛋白直接相互作用进行整合的关键调节剂。目标 1：我们将使用我们新颖且创新的体内整合测定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。在该系统中，广泛表征的小鼠乳腺肿瘤病毒长末端重复序列 (MMTV LTR) 的转录起始是响应类固醇激素而发生的，我们将利用这一特征来绘制有或没有激素的 MoMLV 整合事件图谱。目标 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 筛选中鉴定了许多整合酶相互作用的宿主因子，并且我们将在哺乳动物细胞系中使用 RNA 干扰 (RNAi) 来最初敲低两种蛋白质，这两种蛋白质都是染色质结合因子。使用这项技术，我们证明其中一种蛋白质可以抑制病毒的感染性。我们还将在这些细胞系中存在和不存在激素的情况下将整合事件映射到嵌合牛乳头状瘤病毒-MMTV LTR附加体中。这些研究将通过识别至少两个关键宿主因子并提供体内整合系统来推进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：识别对整合至关重要的宿主因素非常重要，原因有两个。首先，设计良性的基于逆转录病毒的基因靶向载体以避免插入突变。其次，这些信息对于设计抗艾滋病毒、抗癌药物和其他抗逆转录病毒药物非常有用。