Respiratory Syncytial Virus Polymerase Protein and Promoter Interactions

描述(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，也是免疫功能低下患者和老年人疾病的重要原因。此外，RSV与埃博拉、尼帕等其他高致病性病毒关系密切，是研究其分子生物学的良好模型。RSV有一个由病毒聚合酶转录和复制的RNA基因组，以产生mRNA和后代基因组。这个项目的重点是描述RSV聚合酶复合体与其在RSV基因组中的启动子之间的相互作用。这将通过解决三个主要目标来实现。(1)利用表面残基修饰和紫外光交联技术确定RSV聚合酶与启动子中结合位点相互作用的区域。相互作用部位的鉴定将通过反向遗传学确认，使用微型基因组分析，允许分析RSV在细胞内环境中的转录和复制。(2)研究了RNA复制延伸阶段所必需的包膜起始机制。RSV的全长克隆中的包膜信号将发生突变，并将应用正向遗传学方法来确定它可能与哪种病毒蛋白以及该蛋白中的哪个区域相互作用。(3)与聚合酶复合体相互作用的病毒和细胞蛋白将通过聚合酶复合体的免疫沉淀、质谱分析和Western blotting进行鉴定。相关细胞蛋白在RSV RNA合成的不同阶段的作用将通过抑制它们的表达和检测微型基因组系统和RSV感染细胞中的RSV RNA合成来确定。已知的差异调节转录和复制的启动子突变的影响将被研究，以确定它们是否影响结合启动子的聚合酶复合体的组成。这项研究将提供有关蛋白质-RNA复合体的组成和调节的信息，该复合体在RSV的增殖中起关键作用。从长远来看，这项工作将使人们能够对这种复合体进行详细的结构分析，这可能有助于设计控制RSV感染的抑制剂，并可以确定病毒基因组中可能产生的突变，以产生减毒活疫苗。公共卫生相关性：呼吸道合胞病毒(RSV)是一种主要的人类病原体，导致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增殖周期中关键步骤的分子理解，长期目标是开发抗病毒药物和疫苗来控制RSV疾病。

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.