Regulation of mRNA Export by Viral Proteins

描述（由申请人提供）：mRNA从细胞核到细胞质的运输对于基因表达至关重要，并且受到病原体和信号传导途径的高度调节。为了离开细胞核，大多数mRNA通过衔接蛋白（如E1 B-AP 5）与受体NXF 1-p15（TAP-p15）相互作用。另一种mRNA输出因子是Rae 1，它有助于将mRNA对接到核孔复合物蛋白（如核孔蛋白Nup 98）上。然后mRNA输出复合物通过核孔复合物易位到细胞质。我们已经报道了病毒蛋白与mRNA输出途径的组分的相互作用，其诱导mRNA输出的抑制，并提供关于mRNA输出的调节机制的见解。我们发现水泡性口炎病毒（VSV）基质（M）蛋白结合Rae 1，流感病毒的非结构蛋白1（NS 1）与NXF 1-p15、Rae 1和E1 B-1AP 5相互作用。NS 1是A型流感病毒的主要毒力因子，对发病至关重要。这些病毒-宿主相互作用抑制抗病毒蛋白的表达。然而，Nup 98和Rae 1被干扰素（IFN）上调，这构成了一种抗病毒反应机制，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蛋白是Nup 96，它受IFN调节，反过来优先促进IFN调节的mRNA的表达。使用病毒蛋白作为工具，我们建议揭示新的mRNA输出的分子机制。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NS 1和VSV M蛋白与不同形式的mRNA输出复合物相互作用。我们还确定了新的复杂的成分。将使用生物化学方法在体外组装这些mRNA输出复合物。将使用敲低、过表达和诱变策略结合mRNA输出测定进行功能研究。2.研究病毒介导的mRNA输出阻断剂对mRNA输出的调节作用。我们已经确定了流感病毒NS 1蛋白的新型化学抑制剂，其中一些靶向mRNA输出机制的成分。这些抑制剂和mRNA输出机制之间的相互作用的生化分析将进行调查的关键监管机制。还将在体外和体内进行功能测定，以评估这些抑制剂对mRNA输出的影响。3.确定mRNA输出在抗病毒反应中的作用。我们已经报道了Nup 96被IFN上调，并参与抗病毒反应，以促进IFN调节的mRNA的mRNA输出。Nup 96与Sec 13和Seh 1相互作用。为了研究这些Nups在核转运和Nup 96介导的mRNA输出调节和IFN应答之间的关系，我们已经产生了新的亚型小鼠和细胞，其允许单独或与Nup 96组合的Sec 13和Seh 1逐渐下调。总之，这些研究将产生新的信息，mRNA输出的基本机制，以及病毒和宿主如何调节这种机制，以自己的优势。公共卫生相关性：细胞中两个主要区室（细胞核和细胞质）之间的分子运输涉及病毒靶向的过程，因为它们对于抗病毒防御很重要。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.