Role of cis & trans factors in dengue virus translation

顺式的作用

• 批准号: 7247872
• 负责人: Eva Harris
• 金额: $ 29.28万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7247872
• 关键词: 3' Untranslated Regions; Address; Affinity Chromatography; Animal Viruses; Antibodies; Arthropods; Back; Binding Sites; Categories; Cells; Culicidae; Dengue Virus; Enhancers; Flavivirus; Gene Proteins; Genome; Goals; Human; In Vitro; Indium; Length; Maps; Mediating; Methods; Modeling; Mutation; Pharmaceutical Preparations; Poly A; Polymerase; Polyribosomes; Process; Protein Biosynthesis; Proteins; RNA; RNA chemical synthesis; Recombinant Proteins; Recruitment Activity; Replicon; Reporter; Reporting; Research; Research Personnel; Role; Sequence Analysis; Site-Directed Mutagenesis; Small Interfering RNA; Staging; Structure; System; Translations; Untranslated Regions; Viral; Viral Proteins; Virus; Virus Diseases; Work; inhibitor/antagonist; insight; pathogen; programs; translation factor; vaccine development; viral RNA

DESCRIPTION (provided by applicant): Dengue virus (DEN), a Category A priority pathogen, causes the most prevalent arthropod-borne viral illnesses in humans worldwide. Like other viruses, DEN lacks its own translational machinery and thus is dependent on the host cell for translation of viral RNA. DEN and other flaviviruses have a capped, nonpolyadenylated, positive-sense RNA genome. In contrast to many animal viruses, flaviviruses compete efficiently for the cellular translation apparatus without shutting off host protein synthesis. Our long-term goal is to define the mechanism of flavivirus translation, using DEN as a model, and to characterize the viral and cellular factors involved. Specifically, the proposed research investigates the sequence and structural determinants in the DEN 3' untranslated region (UTR) that stimulate viral translation and the factors that mediate this process. We have shown that the 3'UTR of the DEN genome stimulates translation in a manner similar to that reported for cellular and other viral systems but not yet for flaviviruses. In addition, we have identified sequence elements in the 3'UTR that modulate translation efficiency. The DEN 3'UTR can stimulate translation in the absence of viral proteins, leading to the hypothesis that the DEN 3'UTR stimulates viral translation by recruiting cellular factors. This will be addressed via the following two specific aims: Specific Aim 1: Characterize the role of the 3'UTR in stimulation of DEN translation and identify the sequences and structures in the DEN 3'UTR critical for viral translation. We have shown that the DEN 3'UTR acts as a translational enhancer in reporter constructs in both transfected cells and in vitro translation extracts and have begun to analyze sequences that contribute to this effect. Specific Aim 1 will focus on further delineation of sequences and structures in the DEN type 2 (DEN2) 3'UTR that regulate translation in the context of reporter constructs, DEN2 replicons and a DEN2 infectious clone. Specific Aim 2: Identify and characterize proteins that mediate 3'UTR-stimulated DEN translation. This aim focuses on isolation, identification, and characterization of the proteins that mediate viral translation via interaction with the DEN 3'UTR to begin to understand their mechanism of action. This work will provide insight into translation of DEN and other flaviviruses as well as into eukaryotic translational control. Moreover, these studies should identify targets for rational drug and vaccine development of this medically important pathogen.

描述（由申请人提供）：登革热病毒（DEN）是一种优先病原体，导致全球人类中最普遍的节肢动物传播病毒疾病。像其他病毒一样，Den缺乏自己的翻译机制，因此取决于宿主细胞的病毒RNA翻译。 DEN和其他黄素病毒具有限定的，非多聚腺苷酸化的阳性RNA基因组。与许多动物病毒相反，黄病毒在不关闭宿主蛋白质合成的情况下有效地竞争细胞翻译设备。我们的长期目标是使用DEN作为模型来定义黄病毒翻译的机制，并表征涉及的病毒和细胞因素。具体而言，拟议的研究调查了刺激病毒翻译和介导该过程的因素的DEN 3'未翻译区域（UTR）中的序列和结构决定因素。我们已经表明，DEN基因组的3'UTR以类似于细胞和其他病毒系统的方式刺激翻译，但尚未针对黄病毒。此外，我们已经确定了调节翻译效率的3'UTR中的序列元素。在没有病毒蛋白的情况下，DEN 3'UTR可以刺激翻译，从而假设DEN 3'UTR通过募集细胞因子刺激病毒翻译。这将通过以下两个具体目标解决： 特定目的1：表征3'UTR在刺激DEN翻译中的作用，并识别DEN 3'UTR中对病毒翻译至关重要的序列和结构。我们已经表明，DEN 3'UTR充当转染细胞和体外翻译提取物中报告构建体的转化增强剂，并已开始分析有助于这种效果的序列。具体目标1将集中于在DEN类型2（DEN2）3'UTR中进一步描述，该序列和结构在记者构造，DEN2复制子和DEN2感染性克隆的背景下调节翻译。 特定目的2：识别和表征介导3'UTR刺激的DEN翻译的蛋白质。该目标侧重于通过与DEN 3'UTR相互作用开始理解其作用机理的蛋白质的隔离，鉴定和表征。这项工作将洞悉DEN和其他黄病毒的翻译以及真核翻译控制。此外，这些研究应确定这种医学重要病原体的理性药物和疫苗开发的靶标。