Splicing and Nuclear Transport of Influenza Virus mRNA

摘要 细胞核被划分为称为核体的区域，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，或者在某些情况下被破坏。 其它疾病然而，关于核体的结构和功能的知识有限。 引人注目的是，流感病毒破坏了核斑点，一个涉及RNA的核内区室 加工，以剪接病毒M1 mRNA产生M2 mRNA。未剪接的M1 mRNA片段 产生M1基质蛋白，而去除M1转录物中的内部内含子导致M2 mRNA的形式，其编码离子通道。M1和M2蛋白对于病毒运输至关重要 因此，M1至M2 mRNA的核斑点相关剪接是病毒感染的关键方面。 生命周期最近研究表明，细胞蛋白NS 1-BP和hnRNP K形成复合物， 介导M1 mRNA剪接并特异性产生M2 mRNA。重要的是，NS 1-BP或 hnRNP K干扰了M1/M2 mRNA与核斑点的结合，同时破坏了核斑点的结合。 完整性阻碍了M1到M2的剪接。此外，与NS 1-BP结合的流感毒力蛋白NS 1， 还促进M1散斑定位和拼接。相比之下，通过消耗蛋白质来抑制散斑功能， 核心斑点蛋白SON抑制M2产生和病毒复制。因此，M1 mRNA的剪接， M2 mRNA与核斑点直接相关。由于核斑点通常不是 剪接，但剪接因子的储存位点，在核斑点M1到M2剪接代表了一个新的 核内运输途径，这可能代表了抗病毒治疗的新机会。这项建议 利用涉及细胞生物学、RNA生物化学、病毒学和结构生物学的多管齐下的方法， 为了确定NS 1-BP、hnRNP K和NS 1，以及可能的其他蛋白质， 调节核运输并促进斑点处的前体mRNA剪接。高分辨率和活细胞成像 将用于确定介导核转运和斑点的蛋白质因子和RNA序列 流感病毒M1/M2 RNA的定位。将进行平行研究，以确定同样的影响。 RNA序列对NS 1、NS 1-BP和hnRNP K与M1 RNA结合的影响，以及这些影响 序列和相关蛋白质对M1至M2剪接的影响。基因组方法也将用于 确定宿主基因的一个子集是否通过与正常或流感中的M1相似的途径剪接， 被感染的细胞最后，在原子水平上详细研究了NS 1-BP与NS 1和hnRNP K的相互作用 通过单个结构域和蛋白质复合物的结晶。这些研究将共同揭示新的 选择性剪接和核转运之间的联系，以及这些机制如何 流感病毒破坏了这些过程。因此，这里描述的研究将揭示宿主 流感病毒针对的脆弱性，可以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.