title: CDK9-dependent transcriptional elongation in the innate interferon-stimulated gene response to respiratory syncytial virus infection in airway epithelial cells.
abstract: Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections. During infection, the presence of double-stranded RNA (dsRNA) activates the interferon (IFN) regulatory factor 3 (IRF3) transcription factor, an event triggering expression of immediate early, IFN-stimulated genes (ISGs). We examine the role of transcriptional elongation in control of IRF3-dependent ISG expression. RSV infection induces ISG54, ISG56, and CIG5 gene expression in an IRF3-dependent manner demonstrated by IRF3 small interfering RNA (siRNA) silencing in both A549 epithelial cells and IRF3(-/-) MEFs. ISG expression was mediated by the recruitment of IRF3, CDK9, polymerase II (Pol II), and phospho-Ser(2) carboxy-terminal domain (CTD) Pol II to the IFN-stimulated response element (ISRE) binding sites of the IRF3-dependent ISG promoters in native chromatin. We find that RSV infection enhances the activated fraction of cyclin-dependent kinase 9 (CDK9) by promoting its association with bromodomain 4 (BRD4) and disrupting its association with the inhibitory 7SK small nuclear RNA. The requirement of CDK9 activity for ISG expression was shown by siRNA-mediated silencing of CDK9 and by a selective CDK9 inhibitor in A549 cells. In contrast, RSV-induced beta interferon (IFN-β) expression is not influenced by CDK9 inhibition. Using transcript-selective quantitative real-time reverse transcription-PCR (Q-RT-PCR) assays for the ISG54 gene, we observed that RSV induces transition from short to fully spliced mRNA transcripts and that this transition is blocked by CDK9 inhibition in both A549 and primary human small airway epithelial cells. These data indicate that transcription elongation plays a major role in RSV-induced ISG expression and is mediated by IRF3-dependent recruitment of activated CDK9. CDK9 activity may be a target for immunomodulation in RSV-induced lung disease.
authors.affiliation: Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Tian
authors.forename: Yingxin
authors.initials: Y
authors.lastname: Zhao
authors.forename: Mridul
authors.initials: M
authors.lastname: Kalita
authors.forename: Chukwudi B
authors.initials: CB
authors.lastname: Edeh
authors.forename: Slobodan
authors.initials: S
authors.lastname: Paessler
authors.forename: Antonella
authors.initials: A
authors.lastname: Casola
authors.forename: Michael N
authors.initials: MN
authors.lastname: Teng
authors.forename: Roberto P
authors.initials: RP
authors.lastname: Garofalo
authors.forename: Allan R
authors.initials: AR
authors.lastname: Brasier
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism*; D004847:Epithelial Cells / Q000276:immunology / Q000378:metabolism / Q000821:virology*; D006801:Humans; D007113:Immunity, Innate; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000378:metabolism*; D007372:Interferons / Q000378:metabolism*; D008168:Lung / Q000166:cytology / Q000276:immunology / Q000821:virology*; D016601:RNA-Binding Proteins; D018357:Respiratory Syncytial Virus Infections / Q000276:immunology* / Q000821:virology; D018113:Respiratory Syncytial Virus, Human / Q000276:immunology / Q000472:pathogenicity*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C484290:IFIT1 protein, human; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D007372:Interferons; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9