Long Noncoding RNA Lnc-MxA Inhibits Beta Interferon Transcription by Forming RNA-DNA Triplexes at Its Promoter
Long Noncoding RNA Lnc-MxA Inhibits Beta Interferon Transcription by Forming RNA-DNA Triplexes at Its Promoter
复制标题
DOI：
10.1128/jvi.00786-19
复制
发表时间：
2019-11-01
影响因子：
5.4
通讯作者：
Ye, Xin
中科院分区：
文献类型：
--
作者：
Li, Xinda;Guo, Guijie;Ye, Xin
此前，我们鉴定了一组在甲型流感病毒（IAV）感染的细胞中差异表达的长非编码RNA（lncRNA）。在本研究中，我们关注 lnc-MxA，它在 IAV 感染期间上调。我们发现lnc-MxA的过表达促进IAV的复制，而lnc-MxA的敲低则抑制病毒复制。进一步的研究表明lnc-MxA是一种干扰素刺激基因。然而，lnc-MxA 抑制仙台病毒 (SeV) 和 IAV 诱导的 β 干扰素 (IFN-β) 激活。荧光素酶测定表明，lnc-MxA 在 RIG-I、MAVS、TBK1 或活性 IRF3 (IRF3-5D) 刺激下抑制 IFN-β 报告基因的激活。这些数据表明 lnc-MxA 负向调节 RIG-I 介导的抗病毒免疫反应。染色质免疫沉淀（ChIP）检测显示，lnc-MxA过表达细胞中IFN-β启动子处IRF3和p65的富集显着低于对照细胞，表明lnc-MxA干扰IRF3和p65与IFN-β启动子的结合。通过 RNA 纯化 (ChIRP)、三重下拉和生物层干涉测定法进行的染色质分离表明，lnc-MxA 可以与 IFN-β 启动子结合。此外，电泳迁移率变动分析（EMSA）显示lnc-MxA可以与IFN-g启动子片段形成复合物。这些结果表明，lnc-MxA可以与IFN-β启动子形成三链体，干扰IFN-β转录的激活。通过水泡性口炎病毒（VSV）感染测定，我们证实lnc-MxA可以抑制RIG-I样受体（RLR）介导的抗病毒免疫反应并影响细胞的抗病毒状态。总之，我们发现lnc-MxA是一种干扰素刺激基因（ISG），它通过形成RNA-DNA三链体来负向调节IFN-β的转录。 重要性 IAV可以被宿主免疫系统识别为非自身分子模式，并可以引起免疫反应。然而，流感病毒引起的强烈免疫反应（称为“细胞因子风暴”）也会导致广泛的组织损伤（X. Z. J.Guo 和 P.G.Thomas, SeminImmunopathol 39: 541-550, 2017, https://doi.org/10.1007/s00281-017-0636-y; S. Yokota, Nihon Rinsho 61：1953-1958，2003；I.A.Clark，免疫细胞生物学 85：271-273，2007)。与此同时，宿主细胞免疫反应平衡的详细机制尚不清楚。我们的研究表明，作为 IFN 诱导基因，lnc-MxA 充当抗病毒免疫反应的负调节因子。我们揭示了 lnc-MxA 抑制 IFN-β 转录激活的机制。我们的研究结果表明，作为 ISG，lnc-MxA 在维持免疫稳态的负反馈回路中发挥着重要作用。
Previously, we identified a set of long noncoding RNAs (lncRNAs) that were differentially expressed in influenza A virus (IAV)-infected cells. In this study, we focused on lnc-MxA, which is upregulated during IAV infection. We found that the overexpression of lnc-MxA facilitates the replication of IAV, while the knockdown of lnc-MxA inhibits viral replication. Further studies demonstrated that lnc-MxA is an interferon-stimulated gene. However, lnc-MxA inhibits the Sendai virus (SeV)- and IAV-induced activation of beta interferon (IFN-beta). A luciferase assay indicated that lnc-MxA inhibits the activation of the IFN-beta reporter upon stimulation with RIG-I, MAVS, TBK1, or active IRF3 (IRF3-5D). These data indicated that lnc-MxA negatively regulates the RIG-I-mediated antiviral immune response. A chromatin immunoprecipitation (ChIP) assay showed that the enrichment of IRF3 and p65 at the IFN-beta promoter in lnc-MxA-overexpressing cells was significantly lower than that in control cells, indicating that lnc-MxA interfered with the binding of IRF3 and p65 to the IFN-beta promoter. Chromatin isolation by RNA purification (ChIRP), triplex pulldown, and biolayer interferometry assays indicated that lnc-MxA can bind to the IFN-beta promoter. Furthermore, an electrophoretic mobility shift assay (EMSA) showed that lnc-MxA can form complexes with the IFN-g promoter fragment. These results demonstrated that lnc-MxA can form a triplex with the IFN-beta promoter to interfere with the activation of IFN-beta transcription. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that lnc-MxA can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we revealed that lnc-MxA is an interferon-stimulated gene (ISG) that negatively regulates the transcription of IFN-beta by forming an RNA-DNA triplex.IMPORTANCE IAV can be recognized as a nonself molecular pattern by host immune systems and can cause immune responses. However, the intense immune response induced by influenza virus, known as a "cytokine storm," can also cause widespread tissue damage (X. Z. J. Guo and P. G. Thomas, Semin Immunopathol 39: 541-550, 2017, https://doi.org/10.1007/s00281-017-0636-y; S. Yokota, Nihon Rinsho 61:1953-1958, 2003; I. A. Clark, Immunol Cell Biol 85:271-273, 2007). Meanwhile, the detailed mechanisms involved in the balancing of immune responses in host cells are not well understood. Our studies reveal that, as an IFN-inducible gene, lnc-MxA functions as a negative regulator of the antiviral immune response. We uncovered the mechanism by which lnc-MxA inhibits the activation of IFN-beta transcription. Our findings demonstrate that, as an ISG, lnc-MxA plays an important role in the negative-feedback loop involved in maintaining immune homeostasis.