Splicing and Nuclear Transport of Influenza Virus mRNA

流感病毒 mRNA 的剪接和核转运

• 批准号: 9913442
• 负责人: Yuh Min Chook
• 金额: $ 51.79万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-25 至 2021-09-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9913442
• 关键词: Affinity; Affinity Chromatography; Alternative Splicing; Amino Acid Sequence; Antiviral Agents; Antiviral Response; Antiviral Therapy; Binding; Biochemical; Biological Assay; Cell Nucleus; Cells; Cellular biology; Complex; Crystallization; Cytoplasm; Data; Disease; Economic Burden; Excision; Gene Expression; Genes; Genetic; Genomic approach; Goals; Health; Heterogeneous-Nuclear Ribonucleoprotein K; Human; Individual; Infection; Influenza; Influenza A virus; Introns; Ion Channel; Knowledge; Life Cycle Stages; M2 protein; Mediating; Mediator of activation protein; Messenger RNA; Molecular; Molecular Structure; Mutation; Nuclear; Nuclear Structure; Pathway interactions; Process; Production; Proteins; RNA; RNA Biochemistry; RNA Processing; RNA Sequences; RNA Splicing; Regulation; Resolution; Site; Spliced Genes; Structure; Sum; Therapeutic; Transcript; Transport Process; United States; Viral; Viral Genes; Viral Genome; Viral Proteins; Virus; Virus Diseases; Virus Replication; base; biophysical properties; crosslink; experimental study; health economics; human pathogen; imaging study; influenza virulence; influenzavirus; insight; interest; live cell imaging; mRNA Precursor; novel; novel therapeutics; nucleocytoplasmic transport; protein complex; recruit; structural biology; success; trafficking; virology

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.

抽象的 将细胞核分为称为核体的域，这些核体可以正确地坐标 各种基因表达途径。这些途径通常是人类病原体的目标或中断的 其他疾病。但是，关于核体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，核斑点是参与RNA的核内室 处理，以将病毒M1 mRNA拼写成产生M2 mRNA。未填充的M1 mRNA段 生成M1基质蛋白 mRNA的形式，该形式编码一个离子通道。 M1和M2蛋白均对病毒运输至关重要 和萌芽，因此，M1与M2 mRNA的核斑点相关的剪接是病毒的关键方面 生命周期。最近已经显示，细胞蛋白NS1-BP和HNRNP K形成了一个复合物 介导M1 mRNA剪接，并特别产生M2 mRNA。重要的是，NS1-BP或 HNRNP K渗透了M1/M2 mRNA与核斑点的关联，而斑点的破坏 完整性阻碍M1到M2剪接。此外，与NS1-BP结合的流感毒力蛋白NS1 还促进M1斑点定位和剪接。相比之下，通过耗竭抑制斑点功能 核心斑点蛋白儿子抑制M2的产生和病毒复制。因此，M1 mRNA的剪接到 M2 mRNA与核斑点直接相关。由于核斑点通常不是 拼接但是剪接因子的存储位点，M1至M2在核斑点上剪接代表一个新的 核内贩运途径可能代表了抗病毒治疗的新机会。这个建议 利用一种涉及细胞生物学，RNA生物化学，病毒学和结构生物学的多管齐下方法 确定NS1-BP，HNRNP K和NS1的机制，以及其他蛋白质， 调节核贩运并在斑点上促进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M1/M2 RNA的定位。将进行平行研究以确定相同的影响 NS1，NS1-BP和HNRNP K与M1 RNA的结合上的RNA序列，这些序列的影响 M1上的序列和相关蛋白与M2剪接。基因组方法也将用于 确定宿主基因的子集是否在正常或流感中与M1相似的途径剪接 感染细胞。最后，NS1-BP与NS1和HNRNP K的相互作用将以原子级的细节进行研究 通过各个结构域和蛋白质复合物的结晶。这些研究将共同​​发现新颖 替代剪接与核运输之间的机制以及连接以及这些机制 过程被流感病毒颠覆。因此，此处描述的研究将揭示主机 流感病毒瞄准的漏洞有可能用于设计新的治疗选择。