Long Noncoding RNA Lnc-MxA Inhibits Beta Interferon Transcription by Forming RNA-DNA Triplexes at Its Promoter

Abstract

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-β). A luciferase assay indicated that lnc-MxA inhibits the activation of the IFN-β 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-β 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-β promoter. Chromatin isolation by RNA purification (ChIRP), triplex pulldown, and biolayer interferometry assays indicated that lnc-MxA can bind to the IFN-β promoter. Furthermore, an electrophoretic mobility shift assay (EMSA) showed that lnc-MxA can form complexes with the IFN-β promoter fragment. These results demonstrated that lnc-MxA can form a triplex with the IFN-β promoter to interfere with the activation of IFN-β 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-β 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-β transcription. Our findings demonstrate that, as an ISG, lnc-MxA plays an important role in the negative-feedback loop involved in maintaining immune homeostasis.

Of Keeping and Tipping the Balance: Host Regulation and Viral Modulation of IRF3-Dependent IFNB1 Expression

“…LYAR interacts specifically with the N-terminal domain of activated IRF3 and in this way interferes with the DNA-binding capacity of IRF3 in the IRF3-CBP/p300 holocomplex. Additionally, a long non-coding RNA directly targets the IFNβ promoter region and interferes with transcription factor binding in a unique way [ 165 ]: After RNA deep sequencing revealed enhanced transcription of the long non-coding RNA lnc-MxA from the MxA locus after viral infection, Li and colleagues recently reported its mechanism of action. Applying a chromatin isolation by RNA purification assay and in vitro pulldown of an IFNβ promoter dsDNA fragment with biotin-labelled lnc-MxA, they demonstrated that lnc-MxA forms an RNA-DNA triplex with the IFNβ promoter region.…”

“…Applying a chromatin isolation by RNA purification assay and in vitro pulldown of an IFNβ promoter dsDNA fragment with biotin-labelled lnc-MxA, they demonstrated that lnc-MxA forms an RNA-DNA triplex with the IFNβ promoter region. This changes the structure of the chromatin and interferes with binding of the transcription factors IRF3 and NF-κB to their respective target sequence [ 165 ]. Finally, the delayed generation of PRDI-binding factor 1 (PRDI-BF1, or PR/SET domain 1) mediates recruitment of the histone H3 lysine methyltransferase G9a for epigenetic silencing of IFNB1 expression [ 166 , 167 ].…”

Anemoside B4 protects against Klebsiella pneumoniae- and influenza virus FM1-induced pneumonia via the TLR4/Myd88 signaling pathway in mice

“…Influenza virus FM1-infected pneumonia leads to an inflammatory storm, suggesting that many pro-inflammatory cytokines like TNF-α and IL-6 released into blood and lung tissue [28][29][30]. In our study, FM1 was non-invasive intratracheally instilled into lung tissue of mice.…”

LncRNAs in HCV Infection and HCV-Related Liver Disease

“…In turn, lnc-Lsm3b (lncRNA U6 snRNA-associated Sm-like protein) is an IFN-induced lncRNA that blocks the shift in the conformation of RIG-I, required for signaling, limiting IFNβ production [89]. Acting downstream, lnc-MxA (lncRNA Interferon-induced GTP-binding protein MxA) is also an IFN-induced lncRNA that interferes with IFNβ transcription by forming a DNA-RNA triplex helix at the IFNβ promoter [90]. Therefore, lnc-Lsm3b and lnc-MxA are IFN-induced lncRNAs that help viral replication.…”