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.

流感病毒是流感的病因，每年导致数千人死亡。 将来也有一次又一次发生大流行爆发的风险。高水平 循环中的影响力A病毒中对当前抗病毒药物的抗性，新阶级必须 发现在病毒生命周期中针对其他基本步骤的药物。流感病毒 取决于宿主翻译的机械以在感染细胞中产生病毒蛋白。先前的研究已有 表明影响力病毒能够在受帮助的情况下上调感染细胞中病毒蛋白的产生 非结构蛋白1（NS1） NS1是一种26 kDa病毒蛋白，与双链和单链结合 滞留的RNA。 NS1具有两个功能域：RNA结合域（RBD）和效应域（ED）。这 NS1的主要功能是通过与几种宿主蛋白​​相互作用来减少细胞中的干扰-β反应。 此外，已经提出了NS1通过与poly（a）相互作用来刺激病毒mRNA的翻译 结合蛋白1（PABP1）和真核起始因子4G（EIF4G）。但是，使用的机制 NS1专门刺激病毒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 序列。在初步研究中，我们确定了NS1和 PABP1使用荧光各向异性结合了几个RNA。如预期的那样，PABP1具有高结合 与poly（a）RNA的亲和力，但不对poly（c）RNA。有趣的是，我们的研究表明，NS1与双重结合 滞留的RNA，但不构成来自5'-非翻译区域（5'-UTR）的单个链RNA序列 病毒mRNA。我们计划鉴定使用A的病毒mRNA中的RNA基序 定量结合测定和RNA化学探测实验的组合。另外， NS1与PABP1和EIF4G的相互作用将使用Förster共振能量转移（FRET）分析 测定。这些研究将揭示NS1是否与特定的RNA基序结合以募集PABP1和EIF4G至 刺激病毒mRNA的翻译。了解NS1刺激病毒mRNA的机制 翻译将提供新的途径，以防止感染或降低疾病的严重程度。