MECHANISM OF ROTAVIRUS GENOME REPLICATION AND PACKAGING

轮状病毒基因组复制和包装机制

• 批准号: 6431666
• 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/6431666
• 关键词: DNA directed DNA polymerase; Rotavirus; biological signal transduction; capsid; gel electrophoresis; gene mutation; genetic transcription; genetic translation; genome; molecular cloning; polymerase chain reaction; protein biosynthesis; virus genetics; virus replication

Rotaviruses are the most important cause of severe dehydrating diarrhea in infants and young children. The mortality due to rotavirus infection is significant, globally causing nearly 1 million deaths each year. Recently, a vaccine was developed in the Laboratory of Infectious Diseases that provided a high level of protection against the severe diarrhea and dehydration associated with rotavirus infection. Because of concerns that the vaccine may infrequently cause intussusception in young children, the vaccine was withdrawn from the US market by the licensee Wyeth-Lederle. Due to the impact of rotavirus infection on human health, an important overall goal of the Laboratory remains the development of new vaccines, the improvement of existing vaccines, and the identification of other methods for preventing and treating rotaviral disease. Accomplishing this goal would be helped by a fuller understanding of the molecular biology of the virus. In particular, by defining events in the replication and packaging of the segmented double-stranded RNA genome of these viruses, we should gain the information necessary to attenuate pathogenic strains of rotavirus and thereby to molecularly engineer new candidate vaccines. The primary focus of this project is to characterize the fundamental events in the replication of the rotavirus genome which then can be used to develop a reverse genetics system for manipulating the genetic information of the virus. Specifically, this project seeks to identify cis-acting signals that are important for the transport, sorting, packaging and replication of rotavirus mRNAs. The project also seeks to characterize the structure and function of those 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 with 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 requirements for initiation of minus strand synthesis by the viral RNA polymerase, (ii) the nature of cis-acting signals in viral mRNAs that promote the synthesis of the dsRNA genome, (iii) the enzymatic activities of the potential RNA packaging protein NSP2, and (iv) the identity of the viral protein responsible for methylation of the 5 prime-caps of rotavirus mRNAs.

轮状病毒是婴儿和幼儿严重脱水腹泻的最重要原因。轮状病毒感染引起的死亡率很大，全球造成近100万人死亡。最近，在传染病实验室开发了一种疫苗，该实验室为与轮状病毒感染相关的严重腹泻和脱水提供了高度保护。由于担心这种疫苗可能很少引起幼儿的肠套，因此被许可人Wyeth-LeDerle从美国市场撤出了疫苗。由于轮状病毒感染对人类健康的影响，实验室的一个重要总体目标仍然是新疫苗的开发，改善现有疫苗以及鉴定用于预防和治疗轮状病病毒疾病的其他方法。实现这一目标将通过对病毒的分子生物学有更深入的了解。特别是，通过定义这些病毒分割的双链RNA基因组的复制和包装中的事件，我们应该获得减轻轮状病毒的致病性菌株的必要信息，从而对分子进行分子进行新的候选疫苗。该项目的主要重点是表征轮状病毒基因组复制中的基本事件，该事件可用于开发一种反向遗传学系统来操纵病毒的遗传信息。具体而言，该项目旨在识别对轮状病毒mRNA的运输，分类，包装和复制至关重要的顺式作用信号。该项目还试图表征这些过程中涉及的那些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）分析与无细胞系统的突变病毒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）的识别性（III）的特性（III）表征，以及（IIII）的特性（III）表征（III）。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。 去年进行的研究提供了（i）通过病毒RNA聚合酶启动减去链合成的要求，（ii）病毒mRNA中顺式作用信号的性质，这些信号的性质促进了dsRNA基因组的合成，（iii）对潜在的rna Nsp2蛋白nsp2和（IIV）的固定性（IIV）的固定性（IIV）的固定性（IIV）的固定性（IIV）的性质（轮状病毒mRNA的素数。