Biology of Animal Nuclear Mononegaviruses

动物核单链病毒生物学

• 批准号: 7681444
• 负责人: Juan C. de la Torre
• 金额: $ 44.44万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-17 至 2010-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7681444
• 关键词: Acute Promyelocytic Leukemia; Affinity Chromatography; Alternative Splicing; Amino Acids; Animals; Apoptosis; Binding; Biochemical; Biochemical Genetics; Biological; Biological Assay; Biological Models; Biology; Borna disease virus; Bornaviridae; Brain Diseases; Cell physiology; Cells; Cellular biology; Central Nervous System Infections; Characteristics; Chromatin; Chronic; Code; Complement; Complex; Cultured Cells; Cytoplasm; Data; Detection; Disease; Dominant-Negative Mutation; Family; Gene Expression; Generations; Genes; Genetic; Genome; Goals; Health; Hormones; Human; Life Cycle Stages; Mass Spectrum Analysis; Mediating; Messenger RNA; Molecular; Mononegavirales; Mutation; N-terminal; Natural Immunity; Neuraxis; Neurologic; Nuclear; Nuclear Structure; Nucleoproteins; Open Reading Frames; Pattern; Play; Polymerase; Positioning Attribute; Production; Protein Isoforms; Proteins; Proteomics; RNA Interference; RNA Splicing; RNA chemical synthesis; RNA replication; Range; Regulation; Research Personnel; Rodent; Role; Screening procedure; Signal Transduction; Small Interfering RNA; Study models; System; Testing; Trans-Activators; Transcription Elongation; Transcriptional Regulation; Viral; Virus; Virus Diseases; Virus Replication; base; brain cell; cofactor; influenzavirus; inhibitor/antagonist; member; neuropsychiatry; novel; particle; polypeptide; positional cloning; prevent; programs; prototype; transcription factor PML; transcription termination; viral RNA

Borna disease virus (BDV) is an important model for the study of viral persistence in the central nervous system (CNS), a subject relevant to health because viruses can contribute to human neurologic disease. BDV is the prototype of a new family of mononegaviruses, and information obtained from studies on BDV might illuminate novel aspects of MNV biology. Our long-term goal is to elucidate the mechanisms that control BDV replication and gene expression as a prerequisite to understand the molecular bases of BDV persistence in the CNS and virally induced disturbances in brain cell functions. The experimental focus of this proposal is to functionally characterize viral and cellular proteins that contribute to the control of replication and gene expression program of BDV. Our specific aims are: 1. Detection and functional characterization of BDV predicted polypeptides p8.3, p8.4 and p165. BDV uses alternative RNA splicing and transcription termination signals to generate three mRNA species that can code for three novel predicted viral polypeptides whose expression and functions in the virus life cycle have not been studied. We will: 1) Determine whether these polypeptides are produced during BDV infection. 2) Characterize their sub-cellular and temporal expression patterns during BDV infection. 3) Examine their roles in the control of BDV RNA replication and gene expression. 2. Examine the roles of BDV p10 and Np38 polypeptides in the life cycle of BDV. We will determine: 1) The mechanisms whereby p10 inhibits viral RNA synthesis. 2) How p10 modulates its inhibitory activity in BDV-infected cells and whether p10 is required for virus propagation. 3) The mechanisms whereby Np38 contributes to optimal BDV polymerase activity, and whether Np38 is required for the generation of infectious virus and its propagation. 3. Biochemical and functional characterization of the BDV polymerase complex-host cell protein interactions. We will investigate the mechanisms and consequences of BDV polymerase complex-hCLE interaction we have identified. We will use Tandem Affinity Purification (TAP) and Mass Spectrometry approaches to identify the full array of BDV polymerase complex interacting partners. As a complementary approach we will use siRNA-based screenings to identify cellular inhibitors of the BDV polymerase complex. Candidate molecules identified by proteomic approaches and RNAi-based screens will be validated using biochemical and genetic approaches. The biological relevance of validated viruscell protein interactions will be examined in the context of the BDV MG rescue system and BDV-infected cells using a combination of biochemical, genetic and cell biology approaches.

伯尔纳病毒病毒（BDV）是研究中枢神经中病毒持久性研究的重要模型 系统（CNS），这是与健康有关的主题，因为病毒可以导致人类​​神经系统疾病。 BDV是新的单核病毒家族的原型，也是从BDV的研究获得的信息 可能会阐明MNV生物学的新颖方面。我们的长期目标是阐明 控制BDV复制和基因表达是了解BDV分子碱基的先决条件 中枢神经系统的持久性和脑细胞功能的病毒引起的障碍。实验重点 该建议是在功能上表征病毒和细胞蛋白，这有助于控制 BDV的复制和基因表达程序。我们的具体目的是： 1。BDV预测多肽P8.3，P8.4和P165的检测和功能表征。 BDV使用替代的RNA剪接和转录终止信号来产生三个mRNA物种 可以为三种新颖的预测病毒多肽编码，它们的表达和功能在病毒寿命中 尚未研究周期。我们将：1）确定在BDV期间是否产生这些多肽 感染。 2）表征其在BDV感染期间其亚细胞和时间表达模式。 3） 检查它们在控制BDV RNA复制和基因表达中的作用。 2。检查BDV P10和NP38多肽在BDV生命周期中的作用。我们将确定： 1）P10抑制病毒RNA合成的机制。 2）P10如何调节其抑制作用 BDV感染细胞的活性以及病毒传播是否需要P10。 3）机制 np38有助于最佳的BDV聚合酶活性，以及​​是否需要NP38 产生传染病及其传播。 3。BDV聚合酶复杂宿主蛋白的生化和功能表征 互动。我们将研究BDV聚合酶复合酶-HCLE的机制和后果 我们已经确定的互动。我们将使用串联亲和力纯化（TAP）和质谱法 确定BDV聚合酶复合酶相互作用伙伴的完整阵列的方法。作为 互补方法我们将使用基于SIRNA的筛选来识别BDV的细胞抑制剂 聚合酶复合物。通过蛋白质组学方法和基于RNAi的筛选确定的候选分子 将使用生化和遗传学方法进行验证。经过验证的病毒病的生物学相关性 蛋白质相互作用将在BDV MG救援系统和BDV感染的背景下进行检查 使用生化，遗传和细胞生物学方法的组合。