Genome-wide analysis of Dengue virus 2 infected cells. Homo sapiens

The goal of this study was to compare the transcriptional profile (RNA-seq) of Dengue virus 2 and mock infected cells at 24 and 36 hours post infection. 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. 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. Overall design: A549 cells where infected with Dengue virus 2 or mock and after 24 and 36 hours post infection mRNA was purified. Then the transcriptional profile of these cells was analyzed using RNA-seq.

本研究旨在对比登革病毒2型（Dengue virus 2）感染细胞与模拟感染细胞在感染后24、36小时的转录组谱（RNA-seq）。登革病毒NS5蛋白（NS5 protein）在感染细胞的细胞质中发挥多种功能，既可介导病毒RNA复制，又可拮抗宿主抗病毒免疫应答。本研究首次揭示了NS5蛋白在细胞核内的全新功能——其可干扰细胞剪接过程。通过对感染细胞开展全局蛋白质组分析并结合功能实验，我们发现NS5蛋白可结合剪接体复合物并调控内源性剪接过程。具体而言，无论是单独存在的NS5蛋白，还是处于病毒感染环境中的NS5蛋白，均可与U5小核糖核蛋白颗粒（U5 snRNP particle）的核心组分CD2BP2及DDX23发生相互作用，改变可变剪接事件的外显子保留/跳过比率，并改变已知抗病毒因子的mRNA异构体丰度。值得注意的是，借助新近开发的生物信息学工具开展全基因组转录组分析后发现，登革病毒感染会导致细胞内含子滞留现象增多；而沉默特定U5组分则可增强病毒复制能力。多项机制研究表明，NS5蛋白与剪接体的结合会降低前mRNA的加工效率，且该过程不依赖于NS5蛋白的酶活性。本研究推测，NS5蛋白与U5小核糖核蛋白结合会劫持剪接装置，从而营造出更利于病毒复制的低限制性环境。实验设计概况：将A549细胞分为登革病毒2型感染组与模拟感染组，分别于感染后24小时及36小时提取纯化mRNA，随后通过RNA-seq技术分析各组细胞的转录组谱。