Interaction of Influenza A virus NS1 protein with PABP1 and eIF4G

甲型流感病毒是流感的病原体，每年导致数千人死亡。 未来还存在一次又一次爆发大流行的风险。具有高水平的 在流行的甲型流感病毒中对当前抗病毒药物的耐药性，重要的是新的类别 发现了针对病毒生命周期其他基本步骤的药物。流感病毒 依赖宿主的翻译机制在受感染的细胞中产生病毒蛋白。之前的研究已经 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)。NS1是一种26 kDa的病毒蛋白，可与双链和单链病毒结合。 搁浅的RNA。NS1有两个功能结构域：RNA结合域(RBD)和效应域(ED)。这个 Ns1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反应。 此外，NS1被认为通过与Poly(A)相互作用来刺激病毒mRNAs的翻译。 结合蛋白1(PABP1)和真核细胞启动因子4G(EIF4G)。然而，使用的机制 NS1对病毒mRNAs的特异性刺激作用尚不清楚。我们建议使用新的、 荧光定量分析NS1与PABP1、eIF4G和病毒RNA的相互作用 序列。在初步研究中，我们测定了NS1和NS1的平衡解离常数(Kd) PABP1利用荧光各向异性与几个RNA结合。正如预期的那样，PABP1以高结合力结合 与聚(A)RNA亲和力强，但不与聚(C)RNA亲和力强。有趣的是，我们的研究表明，NS1结合了一个双- 链RNA，但不是来自5‘-非翻译区(5’-UTR)的保守单链RNA序列 病毒mRNAs。我们计划使用一种新的方法来识别病毒mRNAs中被NS1识别的RNA基序 结合定量结合实验和RNA化学探针实验。此外， NS1与PABP1和eIF4G的相互作用将用Förster共振能量转移(FRET)来分析 化验。这些研究将揭示NS1是否与特定的RNA基序结合以招募PABP1和eIF4G来 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来预防感染或降低疾病的严重程度。

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.