Virulence Factor at the Interface of Viral and Cellular mRNA Nuclear Export

摘要 病毒历来揭示了新的细胞过程，包括mRNA核输出途径。在这里我们 研究最近发现的被流感病毒mRNAs篡夺以调节其核输出的途径。这 这一过程是由病毒NS1蛋白与细胞和其他病毒蛋白相互作用驱动的。NS1是一个主要的 抑制细胞抗病毒反应并促进病毒基因表达的毒力因子。在这份提案中， NS1介导病毒mRNA核输出的分子机制及其作用机制 一种新的病毒mRNA核输出的小分子抑制物将被检测。尤其是，我们出版的 初步的结构-功能数据表明，NS1抑制细胞(宿主)mRNA的输出 通过与异源二聚体受体Nxf1-NXT1直接相互作用，将Nxf1-NXT1基因转移到胞核。 此外，NS1阻止Nxf1-NXT1与核孔复合体对接。然而，NS1对于 核出口至少部分基本流感mRNAs，尽管这些mRNAs也需要Nxf1。在AIM 在目前的提议中，这一矛盾的观察将通过确定分子 调节流感基因核输出的机制。遗传学的结合，单分子RNA- FISH(SmFISH)、体外和基于细胞的结合分析以及病毒微基因组系统将用于探索 NS1如何促进病毒mRNAs的输出。目标2将解决一部小说的目标识别/验证 病毒mRNA核输出的抑制物。它是基于对一种细胞NS1结合蛋白的鉴定 介导病毒mRNAs和部分细胞mRNAs的核输出，而不是大量细胞mRNAs的核输出。这 提供了一种在不影响大量细胞基因表达的情况下针对流感病毒的方法。我们设计了 并进行了高含量、基于图像的化学筛选，并确定了一种新的病毒mRNA输出抑制物 对块状细胞的信使核糖核酸没有明显的活性。这种小分子能抑制病毒复制 无毒浓度。初步数据表明，这种小分子以细胞内的TAOK2激酶为靶标。 目标2的重点将放在靶标确认和确定这种新型抑制剂的潜在额外靶标上 使用基于光交联的化学方法以及功能遗传、生化和 成像方法。通过确定NS1如何影响信使核糖核酸的输出以及如何调控这一过程 通过一个小分子，我们将揭示非规范信使核糖核酸的基本新机制和调控。 出口途径。此外，该化合物可能作为开发抗病毒药物的先导。

Abstract Viruses have historically revealed novel cellular processes, including mRNA nuclear export pathways. Here we study a recently identified pathway usurped by influenza virus mRNAs to mediate their nuclear export. This process is driven by the viral NS1 protein interaction with cellular and other viral proteins. NS1 is a major virulence factor that inhibits cellular antiviral response and promotes viral gene expression. In this proposal, molecular mechanisms of NS1 functions that mediate viral mRNA nuclear export and the mechanism of action of a novel small molecule inhibitor of viral mRNA nuclear export will be examined. In particular, our published and preliminary structure-function data show that NS1 inhibits the export of cellular (host) mRNA from the nucleus to the cytoplasm by interacting directly with the heterodimeric host mRNA export receptor NXF1-NXT1. Moreover, NS1 prevents NXF1-NXT1 docking to the nuclear pore complex. However, NS1 is also critical for nuclear export of at least some of the essential influenza mRNAs, even though these also require NXF1. In Aim 1 of the current proposal, this paradoxical observation will be investigated by determination of the molecular mechanisms that mediate influenza mRNA nuclear export. A combination of genetics, single-molecule RNA- FISH (smFISH), in vitro and cell-based binding assays, and a viral mini-genome system will be used to explore how NS1 promotes the export of viral mRNAs. Aim 2 will address target identification/validation of a novel inhibitor of viral mRNA nuclear export. It is based on the identification of a cellular NS1-binding protein that mediates nuclear export of viral mRNAs and of a subset of cellular mRNAs, but not bulk cellular mRNA. This provided an avenue to target influenza virus without compromising bulk cellular gene expression. We designed and performed a high-content, image-based chemical screen and identified a novel inhibitor of viral mRNA export that exhibited no significant activity towards bulk cellular mRNA. This small molecule inhibits virus replication at non-toxic concentrations. Preliminary data indicate that this small molecule targets the cellular TAOK2 kinase. The focus of Aim 2 will be on target validation and identification of potential additional targets of this novel inhibitor of mRNA export using photo-crosslinking based chemical approaches, and functional genetic, biochemical, and imaging approaches. By determining how NS1 influences mRNA export and how this process can be regulated by a small molecule, we will uncover fundamental new mechanisms and regulation of a noncanonical mRNA export pathway. Additionally, this compound may serve as a lead for developing antivirals.