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

病毒和细胞 mRNA 核输出界面的毒力因子

• 批准号: 10604277
• 负责人: Beatriz MA Fontoura
• 金额: $ 54.69万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-06 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10604277
• 关键词: Affect; Antiviral Agents; Antiviral Response; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biology; Cell Nucleus; Cell physiology; Cells; Cellular biology; Chemicals; Complex; Cytoplasm; Data; Docking; Exhibits; Future; Gene Expression; Genes; Genetic; Genomic approach; Image; Immunity; In Vitro; Infection; Influenza; Influenza A virus; Knock-out; Label; Lead; Life Cycle Stages; Mediating; Messenger RNA; Molecular; Molecular Genetics; Mutagenesis; Nuclear; Nuclear Export; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Pathogenesis; Pathway interactions; Phosphotransferases; Poly(A)+ RNA; Process; Proteins; Publishing; RNA; Recombinants; Regulation; Reporting; Structure; System; Toxic effect; Validation; Viral; Viral Genes; Viral Genome; Viral Proteins; Virulence Factors; Virus; Virus Inhibitors; Virus Replication; antiviral drug development; cellular targeting; design; drug development; imaging approach; influenzavirus; inhibitor; mRNA Export; novel; overexpression; phosphoproteomics; prevent; receptor; single molecule; small molecule; small molecule inhibitor

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.

摘要 历史上，病毒揭示了新的细胞过程，包括mRNA核输出途径。这里我们 研究一个最近被流感病毒mRNAs篡夺的途径，以介导它们的核输出。这 该过程由病毒NS 1蛋白与细胞和其他病毒蛋白的相互作用驱动。NS 1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。在这一提议中， NS 1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剂将被检查。特别是，我们出版的 初步的结构-功能数据表明，NS 1抑制细胞（宿主）mRNA从细胞膜的输出。 通过与异二聚体宿主mRNA输出受体NXF 1-NXT 1直接相互作用，将细胞核转移到细胞质。 此外，NS 1阻止NXF 1-NXT 1与核孔复合物对接。然而，NS 1对于以下方面也至关重要： 至少一些必需的流感mRNA的核输出，即使这些也需要NXF 1。在Aim中 1的目前的建议，这一矛盾的观察将调查的分子测定 调节流感mRNA核输出的机制。基因，单分子RNA- FISH（smFISH）、体外和基于细胞的结合试验以及病毒微型基因组系统将用于探索 NS 1如何促进病毒mRNA的输出。目标2将解决新的目标识别/验证 病毒mRNA核输出抑制剂。它基于对细胞NS 1结合蛋白的鉴定， 介导病毒mRNA和细胞mRNA子集的核输出，但不介导大量细胞mRNA。这 提供了靶向流感病毒而不损害大量细胞基因表达的途径。我们设计 并进行了高含量的基于图像的化学筛选，并确定了一种新的病毒mRNA输出抑制剂， 其对大量细胞mRNA没有表现出显著的活性。这种小分子抑制病毒复制， 无毒浓度。初步数据表明，这种小分子靶向细胞TAOK 2激酶。 目标2的重点将是目标验证和识别这种新型抑制剂的潜在其他目标 使用基于光交联的化学方法进行mRNA输出，以及功能遗传、生化和 成像方法。通过确定NS 1如何影响mRNA输出以及如何调节这一过程， 通过一个小分子，我们将揭示一个非经典mRNA的基本新机制和调控， 出口通道。此外，这种化合物可以作为开发抗病毒药物的先导。