Biology of Animal Nuclear Mononegaviruses

动物核单链病毒生物学

基本信息

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
项目类别：
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

项目摘要

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.

博尔纳病病毒（BDV）是研究中枢神经病毒持久性的重要模型系统（CNS），一个与健康相关的主题，因为病毒会导致人类神经系统疾病。 BDV 是单负病毒新家族的原型，并且从 BDV 研究中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其机制控制 BDV 复制和基因表达是了解 BDV 分子基础的先决条件中枢神经系统的持续存在以及病毒引起的脑细胞功能紊乱。实验重点该提案旨在对有助于控制病毒和细胞蛋白的功能进行表征 BDV的复制和基因表达程序。我们的具体目标是： 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 RNA 剪接和转录终止信号来生成三种 mRNA 种类可以编码三种新的预测病毒多肽，其在病毒生命中的表达和功能循环没研究过。我们将： 1) 确定这些多肽是否是在 BDV 过程中产生的感染。 2) 表征 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 感染细胞中的活性以及病毒繁殖是否需要 p10。 3）机制其中 Np38 有助于最佳 BDV 聚合酶活性，以及 Np38 是否是 BDV 聚合酶活性所必需的传染性病毒的产生及其传播。 3. BDV聚合酶复合物-宿主细胞蛋白的生化和功能表征互动。我们将研究 BDV 聚合酶复合物-hCLE 的机制和后果我们已经确定了相互作用。我们将使用串联亲和纯化 (TAP) 和质谱法识别全系列 BDV 聚合酶复合物相互作用伙伴的方法。作为一个作为补充方法，我们将使用基于 siRNA 的筛选来鉴定 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 的筛选鉴定出候选分子将使用生化和遗传学方法进行验证。经验证的病毒细胞的生物学相关性将在 BDV MG 救援系统和 BDV 感染的背景下检查蛋白质相互作用使用生物化学、遗传和细胞生物学方法相结合的细胞。