Regulation of mRNA Export by Viral Proteins

病毒蛋白对 mRNA 输出的调控

• 批准号: 7924953
• 负责人: Beatriz MA Fontoura
• 金额: $ 26.69万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924953
• 关键词: Adaptor Signaling Protein; Address; Animals; Antiviral Agents; Antiviral Response; Avian Influenza A Virus; Binding; Biochemical; Biological Assay; Cell Death; Cell Nucleus; Cells; Chemicals; Complex; Cytoplasm; Docking; Down-Regulation; Essential Genes; Gene Expression; Goals; Heterogeneous-Nuclear Ribonucleoprotein Group M; Heterogeneous-Nuclear Ribonucleoprotein U; In Vitro; Influenza A virus; Interferons; Laboratory Study; Light; Mediating; Modeling; Molecular; Mus; Mutagenesis; Nonstructural Protein; Nuclear Export; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Partner in relationship; Pathogenesis; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Process; Protein Binding; Proteins; Regulation; Reporting; Role; Signal Pathway; Therapeutic; Toxic effect; Vesicular stomatitis Indiana virus; Vesicular stomatitis virus M protein; Viral; Viral Proteins; Virulence; Virulence Factors; Virus; Work; base; cytokine; design; high throughput screening; in vivo; influenzavirus; inhibitor/antagonist; insight; interest; knock-down; mRNA Export; member; mouse model; multiple myeloma M Protein; novel; nuclear pore complex protein 96; nucleocytoplasmic transport; overexpression; pathogen; public health relevance; receptor; research study; response; tool; trafficking

DESCRIPTION (provided by applicant): Trafficking of mRNAs from the nucleus to the cytoplasm is essential for gene expression and is highly regulated by pathogens and signaling pathways. To exit the nucleus, most mRNAs interact with the receptors NXF1-p15 (TAP-p15) via adaptor proteins, such as E1B-AP5. Another mRNA export factor is Rae1, which aids in docking mRNAs onto nuclear pore complex proteins, such as the nucleoporin Nup98. The mRNA export complex is then translocated through the nuclear pore complex to the cytoplasm. We have reported the interaction of viral proteins with constituents of the mRNA export pathway, which induced inhibition of Mrna export and provided insights on regulatory mechanisms of mRNA export. We showed that the vesicular stomatitis virus (VSV) matrix (M) protein binds Rae1 and that the nonstructural protein 1 (NS1) of influenza virus interacts with NXF1-p15, Rae1, and E1B-1AP5. NS1 is a major virulence factor of influenza A virus that is essential for pathogenesis. These viral-host interactions inhibit expression of antiviral proteins. However, Nup98 and Rae1 are up-regulated by interferons (IFN), which constitute a mechanism of antiviral response that can revert the mRNA export block mediated by these viral proteins. Another nucleoporin involved in antiviral response is Nup96, which is regulated by IFN and in turn preferentially facilitates expression of IFN-regulated mRNAs. Using viral proteins as tools, we propose to uncover novel molecular mechanisms of mRNA export. Our specific aims are: 1. To investigate the mechanisms through which viral proteins disrupt the mRNA export machinery. We have evidence that NS1 and VSV M proteins interact with different forms of the mRNA export complex. We have also identified novel constituents of the complex. Biochemical approaches will be used to assemble these mRNA export complexes in vitro. Functional studies will be carried out using knockdown, overexpression, and mutagenesis strategies in combination with mRNA export assays. 2. To study regulation of mRNA export by antagonists of viral-mediated mRNA export block. We have identified novel chemical inhibitors of the NS1 protein of influenza virus, some of which target constituents of the mRNA export machinery. Biochemical analyses of interactions between these inhibitors and the mRNA export machinery will be performed to investigate key regulatory mechanisms. Functional assays to assess the effect of these inhibitors on mRNA export will also be performed in vitro and in vivo. 3. To determine the role of mRNA export in antiviral response. We have reported that Nup96 is up-regulated by IFN and is involved in antiviral response to facilitate mRNA export of IFN-regulated mRNAs. Nup96 interacts with Sec13 and Seh1. To study the relationship between these Nups in nuclear transport and Nup96-mediated regulation of mRNA export and IFN response, we have generated novel hypomorphic mice and cells that allow gradual down-regulation of Sec13 and Seh1 alone or in combination with Nup96. Altogether, these studies will generate new information on basic mechanisms of mRNA export and on how viruses and host regulate this machinery to their own advantage. PUBLIC HEALTH RELEVANCE: Trafficking of molecules between the two major compartments in the cell, the nucleus and the cytoplasm, involve processes that are targeted by viruses, as they are important for antiviral defense. Our laboratory studies the mechanisms and key players of these trafficking processes. By understanding these mechanisms, we were able to design inhibitors or drugs that work against viral toxicity.

描述(由申请人提供)：mRNAs从细胞核到细胞质的运输对基因表达是必不可少的，并受到病原体和信号通路的高度调控。为了离开细胞核，大多数mRNAs通过接头蛋白与受体Nxf1-p15(TAP-p15)相互作用，如E1B-AP5。另一种信使核糖核酸输出因子是RAe1，它有助于将信使核糖核酸对接到核孔复合体蛋白上，如核孔蛋白Nup98。然后，mRNA输出复合体通过核孔复合体转移到细胞质。我们已经报道了病毒蛋白与信使核糖核酸输出途径成分的相互作用，从而抑制了信使核糖核酸的输出，并对信使核糖核酸输出的调控机制提供了见解。我们发现水疱性口炎病毒(VSV)基质(M)蛋白与RAe1结合，流感病毒的非结构蛋白1(NS1)与Nxf1-p15、RAe1和E1B-1AP5相互作用。NS1是甲型流感病毒的主要毒力因子，在其致病过程中起着至关重要的作用。这些病毒与宿主的相互作用抑制了抗病毒蛋白的表达。然而，Nup98和Rae1被干扰素上调，这构成了一种抗病毒反应的机制，可以逆转这些病毒蛋白介导的mRNA输出阻断。另一个参与抗病毒反应的核孔素是Nup96，它由干扰素调节，反过来优先促进干扰素调节的mRNAs的表达。利用病毒蛋白作为工具，我们建议揭示新的分子机制的信使核糖核酸输出。我们的具体目标是：1.研究病毒蛋白破坏信使核糖核酸输出机制的机制。我们有证据表明，NS1和VSV M蛋白与不同形式的mRNA输出复合体相互作用。我们还鉴定了该复合体的新成分。将使用生化方法在体外组装这些信使核糖核酸输出复合体。功能研究将使用基因敲除、过表达和突变策略，并结合信使核糖核酸输出分析。2.研究病毒介导的信使核糖核酸输出阻断拮抗剂对信使核糖核酸输出的调节。我们已经确定了流感病毒NS1蛋白的新型化学抑制物，其中一些是mRNA输出机制的靶成分。将对这些抑制剂与信使核糖核酸输出机制之间的相互作用进行生化分析，以研究关键的调控机制。此外，还将在体外和体内进行功能分析，以评估这些抑制剂对信使核糖核酸输出的影响。3.确定信使核糖核酸输出在抗病毒反应中的作用。我们已经报道，Nup96被干扰素上调，并参与抗病毒反应，以促进干扰素调节的mRNAs的mRNA输出。Nup96与Sec13和Seh1相互作用。为了研究这些核转运与Nup96介导的mRNA输出和干扰素反应调节的关系，我们创造了新的低形态小鼠和细胞，允许单独或与Nup96联合逐渐下调Sec13和Seh1的表达。总之，这些研究将产生关于信使核糖核酸输出的基本机制以及病毒和宿主如何为了自己的利益而调节这一机制的新信息。与公共卫生相关：在细胞的两个主要隔间--细胞核和细胞质之间的分子运输涉及病毒靶标的过程，因为它们对抗病毒防御很重要。我们的实验室研究了这些贩运过程的机制和主要参与者。通过了解这些机制，我们能够设计出抗病毒毒性的抑制剂或药物。