Structure and Mechanism of Programmed Ribosomal Frameshifting in SARS coronavirus

SARS冠状病毒程序性核糖体移码的结构和机制

• 批准号: 8477378
• 负责人: Victoria Manuel D'Souza
• 金额: $ 35.74万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-07 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8477378
• 关键词: Antiviral Agents; Attention; Base Pairing; Biochemical; Bypass; Calorimetry; Characteristics; Chemicals; Coronavirus; DNA Sequence Rearrangement; Development; Disease; Engineering; Epidemic; Equilibrium; Event; Frequencies; Gene Expression; Genetic; Goals; In Vitro; Infection; Lung diseases; Mediating; Messenger RNA; Methods; Modeling; Molecular Conformation; Murine leukemia virus; Mutagenesis; Nuclear Magnetic Resonance; Nucleotides; Outcome; Pattern; Physiological; Population; Positioning Attribute; Proteins; Protons; RNA; Reading; Reading Frames; Ribonucleic Acid Regulatory Sequences; Ribosomal Frameshifting; Ribosomes; Severe Acute Respiratory Syndrome; Signal Transduction; Solutions; Structure; Terminator Codon; Testing; Titrations; Translating; Translations; Viral; Virus; Work; base; conformer; design; drug discovery; in vivo; insight; mutant; programs; protonation; public health relevance; research study; response; sensor; tool; translation assay

DESCRIPTION (provided by applicant): Coronaviruses (CoV) are associated with severe diseases as demonstrated by the 2003 severe acute respiratory syndrome (SARS)-CoV epidemic. One of the critical steps of infection involves viral mRNA mediated recoding of gene expression; a -1 frameshifting event that occurs during translation. It is this elegant mechanism that allows the ribosome to bypass a stop codon and synthesize viral enzymatic proteins. Furthermore, the frequency by which this event occurs is important for efficient viral infectivity, and is regulated by domains in the translating mRNA (in the case of the SARS-CoV, this domain is a pseudoknot). Although aspects of frameshifting have received considerable attention, an understanding of the RNA structures and structural rearrangements that influence the efficiency is lacking. Our preliminary studies indicate that retroviral gene expression is regulated by a dynamic, proton-driven equilibrium between an active, read-through permissive, and an inactive pseudoknot conformation. This proposal aims to gain a complete structural and mechanistic understanding of the frameshifting frequency in SARS-CoV by combining structural studies with biochemical and in vivo experiments. Our aims will be: (#1) to understand the basis for how the recoding frequency is maintained, (#2) to determine the structures of the mRNA signal in both configurations: permissive and non-permissive for frameshifting and (#3) to engineer structure-guided mutants to test our equilibrium model.

描述（由申请人提供）：冠状病毒（CoV）与严重疾病相关，如2003年严重急性呼吸系统综合征（SARS）-CoV流行所证明的。感染的关键步骤之一涉及病毒mRNA介导的基因表达重编码;翻译过程中发生的移码事件。正是这种精巧的机制使得核糖体绕过终止密码子合成病毒酶蛋白。此外，该事件发生的频率对于有效的病毒感染性是重要的， 并且受翻译mRNA中的结构域调节（在SARS-CoV的情况下，该结构域是假结）。虽然移码的各个方面受到了相当大的关注，但对影响效率的RNA结构和结构重排的理解仍然缺乏。我们的初步研究表明，逆转录病毒基因的表达是由一个动态的，质子驱动的平衡之间的活性，通读许可，和一个非活性的假结构象。本研究旨在通过结合结构研究、生物化学和体内实验，对SARS冠状病毒的移码频率进行全面的结构和机制研究。我们的目标是：（#1）了解如何保持重新编码频率的基础，（#2）确定两种配置中mRNA信号的结构：允许和不允许移码，（#3）设计结构指导突变体以测试我们的平衡模型。