Interaction of Influenza A virus NS1 protein with PABP1 and eIF4G

甲型流感病毒 NS1 蛋白与 PABP1 和 eIF4G 的相互作用

• 批准号: 9243088
• 负责人: SIMPSON JOSEPH
• 金额: $ 7.08万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9243088
• 关键词: 5' Untranslated Regions; Adamantane; Affinity; Antiviral Agents; Binding; Binding Proteins; Biological Assay; Blood Circulation; Cells; Cessation of life; Chemicals; Conserved Sequence; Disease Outbreaks; Dissociation; Double-Stranded RNA; Drug resistance; Effectiveness; Elements; Energy Transfer; Equilibrium; Eukaryotic Initiation Factor-4G; Fluorescence; Fluorescence Anisotropy; Future; Genetic Translation; Goals; Health; Human; Infection prevention; Influenza; Influenza A virus; Interferon-beta; Knowledge; Life Cycle Stages; Measures; Messenger RNA; Methods; Modeling; Neuraminidase inhibitor; Nucleocapsid Proteins; Play; Poly C; Poly(A) Tail; Poly(A)+ RNA; Poly(A)-Binding Proteins; Production; Proteins; RNA; RNA Binding; RNA Folding; RNA Recognition Motif; RNA Sequences; RNA Viruses; Recruitment Activity; Research; Resistance; Risk; Role; Severity of illness; Specificity; Structure; Time; Translations; Viral; Viral Matrix Proteins; Viral Proteins; Virus; Virus Diseases; Virus Replication; base; effective intervention; experimental study; flu; influenza virus vaccine; influenzavirus; insight; interest; novel drug class; pandemic disease; response; seasonal influenza; therapeutic target; viral RNA

Influenza A virus is the causative agent of flu and is responsible for several thousand deaths annually. There is also the risk of pandemic outbreaks occurring again and again in the future. With high level of resistance to current antiviral drugs among the circulating influenza A viruses, it is important that new classes of drugs are discovered that target other fundamental steps in the life cycle of the virus. Influenza virus depends on the host translational machinery to produce viral proteins in infected cells. Previous studies have indicated that influenza virus is able to up-regulate the production of viral proteins in infected cells with the help of Non-Structural Protein 1 (NS1). NS1 is a 26 kDa viral protein that binds to double-stranded and single- stranded RNAs. NS1 has two functional domains: RNA-binding domain (RBD) and Effector Domain (ED). The primary function of NS1 is to reduce the interferon-β response in cells by interacting with several host proteins. Additionally, NS1 has been proposed to stimulate the translation of viral mRNAs by interacting with Poly (A) Binding Protein 1 (PABP1) and eukaryotic Initiation Factor 4G (eIF4G). However, the mechanism used by NS1 to specifically stimulating the translation of viral mRNAs is not clear. We propose to use new, fluorescence-based quantitative methods to analyze the interaction of NS1 with PABP1, eIF4G, and viral RNA sequences. In preliminary studies, we have determined the equilibrium dissociation constant (KD) for NS1 and PABP1 binding to several RNAs using fluorescence anisotropy. As expected, PABP1 binds with high binding affinity to poly(A) RNA, but not to poly(C) RNA. Interestingly, our studies show that NS1 binds to a double- stranded RNA but not to conserved single stranded RNA sequences from the 5'-untranslated region (5'-UTR) of viral mRNAs. We plan to identify the RNA motifs in viral mRNAs that are recognized by NS1 using a combination of quantitative binding assays and RNA chemical probing experiments. Additionally, the interaction of NS1 with PABP1 and eIF4G will be analyzed using Förster Resonance Energy Transfer (FRET) assays. These studies will reveal whether NS1 binds to specific RNA motifs to recruit PABP1 and eIF4G to stimulate the translation of viral mRNAs. Understanding the mechanism used by NS1 to stimulate viral mRNA translation will provide new avenues to prevent infection or lower the severity of the disease.

甲型流感病毒是流感的病原体，每年导致数千人死亡。 未来还存在疫情反复爆发的风险。 由于正在流行的甲型流感病毒对现有抗病毒药物产生耐药性，因此新类别药物的研发非常重要 已发现针对流感病毒生命周期基本步骤的其他药物。 先前的研究表明，病毒蛋白依赖于宿主翻译机制。 表明流感病毒能够在感染细胞的帮助下上调病毒蛋白的产生 非结构蛋白 1 (NS1) 是一种 26 kDa 的病毒蛋白，可结合双链和单链。 NS1 具有两个功能域：RNA 结合域 (RBD) 和效应器域 (ED)。 NS1 的主要功能是通过与多种宿主蛋白​​相互作用来减少细胞中的干扰素-β 反应。 此外，NS1 已被提议通过与 Poly (A) 相互作用来刺激病毒 mRNA 的翻译 然而，结合蛋白 1 (PABP1) 和真核起始因子 4G (eIF4G) 所使用的机制。 NS1 是否能够特异性刺激病毒 mRNA 的翻译尚不清楚，我们建议使用新的、 基于荧光的定量方法分析 NS1 与 PABP1、eIF4G 和病毒 RNA 的相互作用 在初步研究中，我们已经确定了 NS1 和 NS1 的平衡解离常数 (KD)。 PABP1 利用荧光各向异性与多种 RNA 结合 正如预期的那样，PABP1 具有高结合力。 我们的研究表明，NS1 与聚 (A) RNA 具有亲和力，但与聚 (C) RNA 没有亲和力。 链 RNA，但不包括 5'-非翻译区 (5'-UTR) 的保守单链 RNA 序列 我们计划使用 NS1 识别病毒 mRNA 中的 RNA 基序。 定量结合测定和 RNA 化学探测实验的结合。 NS1 与 PABP1 和 eIF4G 的相互作用将使用福斯特共振能量转移 (FRET) 进行分析 这些研究将揭示 NS1 是否与特定 RNA 基序结合以招募 PABP1 和 eIF4G 刺激病毒 mRNA 的翻译 了解 NS1 刺激病毒 mRNA 的机制。 翻译将为预防感染或降低疾病的严重程度提供新途径。