The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing.
The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing.
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DOI：
10.1371/journal.ppat.1005841
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发表时间：
2016-08
期刊：
影响因子：
6.7
通讯作者：
Gamarnik AV
中科院分区：
文献类型：
--
作者：
De Maio FA;Risso G;Iglesias NG;Shah P;Pozzi B;Gebhard LG;Mammi P;Mancini E;Yanovsky MJ;Andino R;Krogan N;Srebrow A;Gamarnik AV
登革病毒NS 5蛋白在感染细胞的细胞质中发挥多种功能，使病毒RNA复制和抵消宿主的抗病毒反应。在这里，我们展示了一个新的功能NS 5在细胞核中，它干扰细胞剪接。利用感染细胞的全局蛋白质组学分析和功能研究，我们发现NS 5结合剪接体复合物并调节内源性剪接以及小基因衍生的选择性剪接模式。特别是，我们表明，单独的NS 5，或在病毒感染的情况下，与U 5 snRNP颗粒，CD 2BP 2和DDX 23的核心成分相互作用，改变选择性剪接事件的包含/排除比，并改变已知的抗病毒因子的mRNA亚型丰度。有趣的是，使用最近开发的生物信息学工具进行的全基因组转录组分析揭示了登革病毒感染后内含子保留的增加，并且通过沉默特定的U 5组分改善了病毒复制。不同的机制研究表明，NS 5与剪接体的结合降低了前mRNA加工的效率，与NS 5酶活性无关。我们认为NS 5与U 5 snRNP蛋白的结合劫持了剪接机制，从而导致病毒复制的限制性较小的环境。绘制宿主-病原体相互作用已被证明是理解病毒如何操纵宿主机制以及感染期间细胞过程如何调节的基础。登革热病毒对公众健康构成重大威胁：世界上三分之二的人口现在面临感染这种蚊媒病毒的风险。在这项工作中，使用全球蛋白质组学方法的背景下，病毒感染的标记登革病毒，我们构建了一个全面的蛋白质-蛋白质相互作用图的多功能NS 5病毒蛋白。NS 5是病毒RNA复制和免疫逃避的核心。我们的研究揭示了NS 5与剪接机制的核心组分的相互作用，特别是与U 5小核核糖核蛋白颗粒的蛋白质的相互作用，并且病毒感染降低了剪接效率。分析内源性剪接事件和体外剪接测定的机制研究表明，NS 5结合活性剪接体并降低前体mRNA加工的效率。我们的研究结果提供了登革病毒NS 5蛋白的新功能，并支持一种模型，其中操纵特定的剪接成分有利于病毒感染。
Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication. Mapping host-pathogen interactions has proven fundamental for understanding how viruses manipulate host machinery and how cellular processes are regulated during infection. Dengue virus poses a major threat to public health: two-thirds of the world’s population is now at risk from infection by this mosquito-borne virus. In this work, using a global proteomic approach in the context of viral infections with tagged dengue viruses, we constructed a comprehensive protein-protein interaction map of the multifunctional NS5 viral protein. NS5 is central for viral RNA replication and for immune evasion. Our studies revealed the interaction of NS5 with core components of the splicing machinery, specifically with proteins of the U5 small nuclear ribonucleoprotein particle, and that viral infection reduces splicing efficiency. Mechanistic studies analyzing endogenous splicing events and in vitro splicing assays indicated that NS5 binds active spliceosomes and reduces the efficiency of pre-mRNA processing. Our results provide a new function of the dengue virus NS5 protein and support a model in which manipulation of specific splicing components favors viral infection.
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