Respiratory Syncytial Virus Polymerase Protein and Promoter Interactions

呼吸道合胞病毒聚合酶蛋白和启动子相互作用

• 批准号: 7910598
• 负责人: Rachel Fearns
• 金额: $ 40.22万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7910598
• 关键词: Address; Affect; Age; Antiviral Agents; Antiviral Therapy; Applications Grants; Attenuated; Attenuated Live Virus Vaccine; Binding; Binding Sites; Biological Assay; Bronchiolitis; Cells; Childhood; Cis-Acting Sequence; Commit; Complex; Controlled Study; Disease; Ebola virus; Elderly; Environment; Event; Gene Expression; Genetic; Genetic Transcription; Genome; Goals; Health; Hospitalization; Human; Human Virus; Human respiratory syncytial virus; Immunocompromised Host; Immunoprecipitation; Individual; Infant; Length; Life; Lung diseases; Mammalian Cell; Maps; Mass Spectrum Analysis; Measles; Mediating; Messenger RNA; Modification; Molecular; Molecular Biology; Mononegavirales; Mumps; Mutation; Plague; Play; Pneumonia; Poly A; Polymerase; Positioning Attribute; Process; Promoter Regions; Proteins; RNA; RNA chemical synthesis; RNA replication; RNA-Directed RNA Polymerase; Rabies virus; Regulation; Relative (related person); Research; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus Vaccines; Respiratory Tract Infections; Respiratory syncytial virus; Role; Series; Signal Transduction; Site; Staging; Study models; Surface; System; Techniques; Transcription Process; Vaccines; Viral Proteins; Virus; Virus Diseases; Virus Replication; Western Blotting; Work; base; crosslink; design; drug development; inhibitor/antagonist; insight; member; pathogen; positional cloning; promoter; public health relevance; viral RNA

DESCRIPTION (provided by applicant): Respiratory syncytial virus (RSV) is the most important cause of respiratory infection in infants and is a significant cause of disease in immunocompromised patients and the elderly. In addition, RSV is closely related to other highly pathogenic viruses, such as Ebola and Nipah, and is a good model for studying their molecular biology. RSV has an RNA genome that is transcribed and replicated by the virus polymerase to produce mRNA and progeny genomes. The focus of this project is the characterization of the interaction of the RSV polymerase complex with its promoter in the RSV genome. This will be achieved by addressing three major aims. (1) The region of the RSV polymerase that interacts with its binding site in the promoter will be identified using surface residue modification and UV cross-linking techniques. Identification of the interacting site will be confirmed by reverse-genetics, using a minigenome assay that allows analysis of RSV transcription and replication in an intracellular environment. (2) The mechanism of encapsidation initiation, necessary for the elongation stage of RNA replication will be examined. Mutations will be made in the encapsidation signal in the full-length clone of RSV and a forward-genetics approach will be applied to identify which virus protein, and which region within that protein, it might interact with. (3) Virus and cellular proteins that interact with the polymerase complex will be identified using immunoprecipitation of polymerase complexes, mass spectrometry, and Western blotting. The roles of associated cellular proteins during different stages of RSV RNA synthesis will be determined by inhibiting their expression and examining RSV RNA synthesis in the minigenome system and RSV infected cells. The effects of promoter mutations that are known to differentially modulate transcription and replication will be investigated to determine if they affect the composition of the polymerase complex that binds the promoter. This research will provide information regarding the composition and regulation of a protein-RNA complex that plays a key role in RSV multiplication. In the long-term, this work will allow detailed structural analysis of this complex to be performed, which could aid in design of inhibitors to control RSV infection, and could identify mutations that could be made in the virus genome to generate a live- attenuated RSV vaccine. PUBLIC HEALTH RELEVANCE: Respiratory syncytial virus (RSV) is a major human pathogen, responsible for significant numbers of pediatric hospitalizations and for seasonal disease in the elderly. The aim of this project is to progress our molecular understanding of a critical step in the virus multiplication cycle, with the long-term goal of developing antiviral drugs and vaccines to control RSV disease.

描述（由申请人提供）：呼吸道合胞病毒（RSV）是婴儿呼吸道感染的最重要原因，是免疫功能低下的患者和老年人造成疾病的重要原因。此外，RSV与其他高度致病性病毒（例如埃博拉病毒和NIPAH）密切相关，并且是研究其分子生物学的良好模型。 RSV的RNA基因组由病毒聚合酶转录和复制以产生mRNA和后代基因组。该项目的重点是RSV聚合酶复合物与RSV基因组中启动子的相互作用的表征。这将通过解决三个主要目标来实现。 （1）将使用表面残基修饰和紫外线交联技术鉴定与启动子中与其结合位点相互作用的RSV聚合酶区域。相互作用位点的识别将通过反苯二苯甲酸证实，使用微型苯甲组测定法，该测定法可以在细胞内环境中分析RSV转录和复制。 （2）将检查封装启动的机理，即RNA复制延伸阶段所必需的。将在RSV的全长克隆中的封装信号中进行突变，并将应用正向遗传学方法来识别哪种病毒蛋白质以及该蛋白质中的哪个区域可能与之相互作用。 （3）将使用聚合酶复合物，质谱和蛋白质印迹的免疫沉淀来鉴定与聚合酶复合物相互作用的病毒和细胞蛋白。相关细胞蛋白在不同阶段的RSV RNA合成中的作用将通过抑制其表达并检查Minigenome系统和RSV感染细胞中的RSV RNA合成来确定。将研究差异调节转录和复制的启动子突变的影响，以确定它们是否影响结合启动子的聚合酶复合物的组成。这项研究将提供有关在RSV乘法中起关键作用的蛋白质RNA复合物的组成和调节的信息。从长远来看，这项工作将允许对该复合物进行详细的结构分析，这可以有助于设计抑制剂以控制RSV感染，并可以鉴定可以在病毒基因组中进行的突变，从而产生生命减弱的RSV疫苗。公共卫生相关性：呼吸道综合病毒（RSV）是主要的人类病原体，负责大量儿科住院和老年人的季节性疾病。该项目的目的是提高我们对病毒繁殖周期中关键步骤的分子理解，其长期目标是开发抗病毒药和疫苗以控制RSV疾病。