Splicing and Nuclear Transport of Influenza Virus mRNA

摘要 核被划分成被称为核体的区域，这些区域用于适当地协调 不同的基因表达途径。这些途径通常是人类病原体的目标或在 其他疾病。然而，关于核机构的结构和功能的知识有限。 令人惊讶的是，流感病毒颠覆了核斑点，核斑点是与RNA有关的核内隔间 加工过程中，将病毒M1mRNA剪接，生成M2mRNA。未剪接的M1基因片段 产生M1基质蛋白，而去除M1转录本中的内部内含子会导致M2 信使核糖核酸的形式，它编码一个离子通道。M1和M2蛋白都是病毒传播所必需的 因此，核斑点相关的M1和M2 mRNA的剪接是病毒的一个关键方面 生命周期。最近的研究表明，细胞蛋白NS1-BP和hnRNP K形成一个复合体，以 介导M1信使核糖核酸的剪接，并特异性地产生M2信使核糖核酸。重要的是，NS1-BP或 HnRNP K干扰M1/M2 mRNAs与核斑点的联系，同时破坏斑点 完整性阻碍了M1到M2的拼接。此外，与NS1-BP结合的流感毒力蛋白NS1， 还可以促进M1斑点定位和拼接。相反，通过耗尽斑点功能抑制斑点功能 核心斑点蛋白SON，抑制M2的产生和病毒复制。因此，M1 mRNA的剪接到 M2基因的表达与核斑点有直接关系。因为核斑点通常不是 剪接但都是剪接因子的存储位置，在核斑点的M1到M2剪接代表了一种新的 核内转运途径，可能代表抗病毒治疗的新机会。这项建议 利用多管齐下的方法，涉及细胞生物学、RNA生物化学、病毒学和结构生物学 为了确定NS1-BP、hnRNP K和NS1，可能还有其他蛋白质， 规范核贩运，促进斑点前信使核糖核酸的剪接。高分辨率活细胞成像 将被用来确定介导核运输和斑点的蛋白质因子和RNA序列 流感M1/M2RNA的定位。将进行平行研究，以确定相同的影响 RNA序列对NS1、NS1-BP和hnRNP K与M1 RNA结合的影响 M1到M2剪接上的序列和相关蛋白质。基因组学方法也将用于 确定宿主基因的一个子集是否通过与正常或流感中的M1相似的途径拼接- 被感染的细胞。最后，我们将在原子水平上详细研究NS1-BP与NS1和hnRNP K的相互作用 通过单个结构域和蛋白质复合体的结晶。这些研究将共同揭示小说 选择性剪接和核运输的机制和之间的联系，以及这些 流感病毒会颠覆这一过程。因此，这里描述的研究将揭示宿主 流感病毒针对的脆弱性，可能被用来设计新的治疗方案。

Abstract The nucleus is compartmentalized into domains termed nuclear bodies, which serve to properly coordinate various gene expression pathways. These pathways are often targeted by human pathogens or disrupted in other diseases. However, there is limited knowledge regarding the structure and function of nuclear bodies. Strikingly, the influenza virus subverts nuclear speckles, an intranuclear compartment involved in RNA processing, to splice the viral M1 mRNA to generate M2 mRNA. The unspliced M1 mRNA segment generates the M1 matrix protein whereas removal of an internal intron in the M1 transcript leads to the M2 form of the mRNA, which encodes an ion channel. Both M1 and M2 proteins are essential for viral trafficking and budding, thus the nuclear speckle-associated splicing of M1 to M2 mRNA is a critical aspect of the viral life cycle. It has recently been shown that the cellular proteins NS1-BP and hnRNP K form a complex to mediate M1 mRNA splicing and specifically yield the M2 mRNA. Importantly, depletion of either NS1-BP or hnRNP K perturbs the association of the M1/M2 mRNAs with nuclear speckles, while disruption of speckle integrity impedes M1 to M2 splicing. Moreover, the influenza virulence protein NS1, which binds to NS1-BP, also promotes M1 speckle localization and splicing. By contrast, inhibition of speckle function by depletion of the core speckle protein SON, inhibits M2 production and viral replication. Thus, the splicing of M1 mRNA to M2 mRNA is directly associated with nuclear speckles. Since nuclear speckles are not usually sites for splicing but are storage sites of splicing factors, the M1 to M2 splicing at nuclear speckles represents a new intranuclear trafficking pathway that may represent a novel opportunity for antiviral therapy. This proposal leverages a multi-pronged approach involving cell biology, RNA biochemistry, virology and structural biology to determine the mechanisms through which NS1-BP, hnRNP K and NS1, and perhaps additional proteins, regulate nuclear trafficking and promote pre-mRNA splicing at speckles. High-resolution and live cell imaging will be used to determine the protein factors and RNA sequences that mediate nuclear transport and speckle localization of the influenza M1/M2 RNAs. Parallel studies will be done to determine the impact of the same RNA sequences on the binding of NS1, NS1-BP and hnRNP K to the M1 RNA, and the impact of these sequences and associated proteins on M1 to M2 splicing. Genomic approaches will also be used to determine if a subset of host genes are spliced by a similar pathway as M1 in either normal or influenza- infected cells. Finally, the interaction of NS1-BP with NS1 and hnRNP K will be studied in atomic-level detail by crystallization of individual domains and protein complexes. Together these studies will uncover novel mechanisms of, and connections between, alternative splicing and nuclear transport and how these processes are subverted by the influenza virus. As such, the studies described here will reveal host vulnerabilities targeted by influenza virus that can potentially be used to devise new therapeutic options.