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，每年造成数千人死亡。 此外，未来还存在大流行疫情反复爆发的风险。水平高 在流行的甲型流感病毒中，对目前的抗病毒药物具有耐药性，重要的是， 针对病毒生命周期中其他基本步骤的药物被发现。流感病毒 依赖于宿主翻译机器在感染细胞中产生病毒蛋白。先前的研究 表明流感病毒能够在感染细胞中上调病毒蛋白的产生， 非结构蛋白1（NS 1）NS 1是一种26 kDa的病毒蛋白，其结合双链和单链抗体。 单链RNA NS 1具有两个功能结构域：RNA结合结构域（RBD）和效应结构域（艾德）。的 NS 1的主要功能是通过与几种宿主蛋白相互作用来降低细胞中的干扰素-β应答。 另外，已经提出NS 1通过与Poly（A）相互作用来刺激病毒mRNA的翻译。 结合蛋白1（PABP 1）和真核起始因子4G（eIF 4G）。然而， NS 1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NS 1与PABP 1、eIF 4G和病毒RNA的相互作用 序列的在初步研究中，我们已经确定了NS 1的平衡解离常数（KD）， 使用荧光各向异性将PABP 1结合到几种RNA。正如预期的那样，PABP 1以高结合力结合 对poly（A）RNA的亲和力，但对poly（C）RNA没有亲和力。有趣的是，我们的研究表明，NS 1结合到一个双- 链RNA，但不与来自5 '-非翻译区（5'-UTR）的保守单链RNA序列连接 病毒的mRNA。我们计划使用一种新的方法来鉴定病毒mRNA中被NS 1识别的RNA基序。 定量结合测定和RNA化学探测实验的组合。另夕h 将使用Förster共振能量转移（FRET）分析NS 1与PABP 1和eIF 4G的相互作用 测定。这些研究将揭示NS 1是否与特定的RNA基序结合以招募PABP 1和eIF 4G， 刺激病毒mRNA的翻译。了解NS 1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的途径。