Regulation of mRNA Export by Viral Proteins

描述（由申请人提供）：mrna从细胞核转运到细胞质对基因表达至关重要，并受到病原体和信号通路的高度调节。为了离开细胞核，大多数mrna通过衔接蛋白（如E1B-AP5）与受体NXF1-p15 （TAP-p15）相互作用。另一个mRNA输出因子是Rae1，它帮助mRNA对接到核孔复合体蛋白上，如核孔蛋白Nup98。mRNA输出复合体随后通过核孔复合体转运到细胞质。我们报道了病毒蛋白与mRNA输出通路成分的相互作用，从而诱导mRNA输出的抑制，并为mRNA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，对其发病至关重要。这些病毒-宿主相互作用抑制抗病毒蛋白的表达。然而，干扰素（IFN）上调Nup98和Rae1，这构成了抗病毒反应的机制，可以逆转由这些病毒蛋白介导的mRNA输出阻断。另一种参与抗病毒反应的核孔蛋白是Nup96，它受IFN调控，进而优先促进IFN调控mrna的表达。以病毒蛋白为工具，我们提出揭示mRNA输出的新分子机制。我们的具体目标是：1。研究病毒蛋白破坏mRNA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输出复合物相互作用。我们还发现了该复合体的新成分。生化方法将用于体外组装这些mRNA输出复合物。功能研究将使用敲低、过表达和诱变策略结合mRNA输出试验进行。2. 目的：研究病毒介导的mRNA输出阻断拮抗剂对mRNA输出的调控作用。我们已经确定了流感病毒NS1蛋白的新型化学抑制剂，其中一些靶向mRNA输出机制的成分。将对这些抑制剂和mRNA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。评估这些抑制剂对mRNA输出影响的功能分析也将在体外和体内进行。3. 目的：探讨mRNA输出在抗病毒应答中的作用。我们已经报道了Nup96被IFN上调，并参与抗病毒反应，促进IFN调控的mRNA输出。Nup96与Sec13和Seh1相互作用。为了研究这些核转运中的Nups与Nup96介导的mRNA输出和IFN反应的调节之间的关系，我们培育了新的半胚小鼠和细胞，允许Sec13和Seh1单独或与Nup96联合逐渐下调。总之，这些研究将产生关于mRNA输出的基本机制以及病毒和宿主如何调节这一机制以发挥自身优势的新信息。公共卫生相关性：分子在细胞的两个主要区室（细胞核和细胞质）之间的运输涉及病毒靶向的过程，因为它们对抗病毒防御很重要。我们的实验室研究这些贩运过程的机制和主要参与者。通过了解这些机制，我们能够设计出对抗病毒毒性的抑制剂或药物。

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.