Mechanism Of Rotavirus Genome Replication And Packaging

轮状病毒基因组复制和包装机制

• 批准号: 7194132
• 负责人: JOHN PATTON
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7194132
• 关键词: RNA binding protein; Rotavirus; X ray crystallography; analytical ultracentrifugation; capsid; cell free system; crosslink; cryoelectron microscopy; diarrhea; gel mobility shift assay; gene mutation; genetic regulatory element; nuclear magnetic resonance spectroscopy; protein structure function; replicase; structural biology; virus RNA; virus genetics; virus protein; virus replication

Rotaviruses are the primary cause of severe dehydrating diarrhea in infants and young children, globally causing an estimated 2 million hospitalizations and 440,000 deaths per year in children under the age of 5. Due to its significant morbidity and mortality, an important goal of the Laboratory of Infectious Diseases remains the development of methods for controlling, preventing, and treating rotaviral disease. Accomplishing this goal would be helped by a more complete understanding of the molecular biology of rotavirus, notably those events in the viral life cycle connected to the replication of the virus's segmented double-stranded (ds)RNA genome. An expected outcome of this project is the development of a reverse genetics system that could be used in manipulating the genetic information of the virus. Such a system would provide an important tool for generating new and modifying existing rotavirus vaccines. This project may also lead to the identification of potential targets for antiviral components that can subvert the rotavirus replication cycle. Specifically, this project seeks to identify and characterize molecular signals in viral RNAs that function in the expression of rotavirus genes and in the packaging and replication of the rotavirus genome. The project also seeks to characterize the structure and function of viral proteins involved in these processes. These aims will be accomplished by a combination of procedures, which include (i) analysis of the replication and translation efficiencies of mutated viral mRNAs in cell-free systems, (ii) computer modeling and structural analysis (e.g., RNAse mapping, NMR spectroscopy) of the recognition signals in the mRNAs, (iii) characterization of the enzymatic and structural properties (e.g., analytical ultracentrifugation, X-ray crystallography, cryo-electron microscopy, CD spectroscopy) of recombinant viral proteins, and (iv) elucidation of the specificity and targets of the viral RNA-binding proteins by gel mobility shift assay and RNA-protein cross-linking. Studies performed in the last year have provided insight into (i) the atomic structure and/or enzymatic properties of NSP2 and NSP5, viral nonstructural proteins required for viroplasm formation and involved in genome replication and packaging, (ii) the atomic structure and enzymatic properties of the viral RNA-dependent RNA polymerase, VP1, including the role of the inner capsid protein, VP2, in the activation of the polymerase, and (iii) the location and function of cis-acting elements in viral RNAs important for polymerase recognition and for genome replication and packaging. These analyses have been particularly significant in understanding the protein-protein and protein-RNA interactions, and their connected activities, that drive the assembly of early replication intermediates in the viroplasms (viral factories) that form in the cytoplasm of rotavirus-infected cells.

轮状病毒是婴儿和幼儿严重脱水腹泻的主要原因，在全球范围内，估计5岁以下儿童每年造成200万个住院和440,000人死亡。由于其显着的发病率和死亡率，由于其显着的发病率和死亡的重要目的，这是感染性疾病实验室的重要目标，仍然是控制方法的发展，可防止和治疗腐烂的方法。实现这一目标将通过对轮状病毒的分子生物学的更全面了解，特别是与病毒生命周期中的那些事件有关，这些事件与病毒分割的双链（DS）RNA基因组相关。该项目的预期结果是可以用于操纵病毒遗传信息的反向遗传系统的发展。这样的系统将为生成新的和修改现有轮状病毒疫苗的重要工具。该项目还可能导致鉴定可以颠覆轮状病毒复制周期的抗病毒成分的潜在靶标。 具体而言，该项目旨在识别和表征病毒RNA中的分子信号，这些信号在轮状病毒基因表达以及轮状病毒基因组的包装和复制中起作用。该项目还旨在表征与这些过程有关的病毒蛋白的结构和功能。这些目标将通过一系列过程来实现，其中包括（i）分析无细胞系统中突变病毒mRNA的复制和翻译效率，（ii）计算机建模和结构分析（例如，RNase映射，NMR光谱）对mRNA的识别信号（（III）的特性（III）表征，（III）的识别信号（III）表征（iiii）。超速离心，X射线晶体学，重组病毒蛋白的冷冻电子显微镜，CD光谱）以及（iv）通过凝胶动弹性移动性转移测定和RNA蛋白质交叉链接阐明病毒RNA结合蛋白的特异性和靶标。 Studies performed in the last year have provided insight into (i) the atomic structure and/or enzymatic properties of NSP2 and NSP5, viral nonstructural proteins required for viroplasm formation and involved in genome replication and packaging, (ii) the atomic structure and enzymatic properties of the viral RNA-dependent RNA polymerase, VP1, including the role of the inner capsid protein, VP2,在聚合酶的激活中，以及（iii）在病毒酶识别以及基因组复制和包装中，顺式作用元件的位置和功能。这些分析在理解蛋白质 - 蛋白质和蛋白-RNA相互作用及其连接活性方面尤其重要，这些蛋白质 - 蛋白-RNA相互作用及其连接活性，这些活性驱动了在反射病毒感染细胞细胞质中形成的病毒肿瘤（病毒工厂）中早期复制中间体的组装。