title: Sensing adenovirus infection: activation of interferon regulatory factor 3 in RAW 264.7 cells.
abstract: We have used the RAW 264.7 murine macrophage-like cell line as a platform to characterize the recognition and early signaling response to recombinant adenoviral vectors (rAdV). Infection of RAW 264.7 cells triggers an early response (2 to 6 h postinfection) that includes phosphorylation of the interferon (IFN) response factor 3 (IRF3) transcription factor, upregulation of IRF3 primary response genes (interferon-stimulated gene 56 [ISG56], beta IFN [IFN-β]), and subsequent type I IFN secondary signaling (STAT1/2 phosphorylation). Using short hairpin RNA (shRNA) lentiviral vectors, we show an essential role for Tank binding kinase 1 (TBK1) in this pathway. Data also support a role for STING (MITA) as an adaptor functioning in response to rAdV infection. Using UV/psoralen (Ps)-inactivated virus to block viral transcription, Ps-inactivated virus stimulated primary (IRF3) and secondary (STAT1/2) activation events to the same degree as untreated virus. IRF3 phosphorylation was not blocked in RAW 264.7 cells pretreated with the RNA polymerase III inhibitor ML60218. However, they were compromised in the type I IFN-dependent secondary response (phosphorylation of STAT1/STAT2). At 24 h postinfection, ML60218-treated cells were compromised in the overall antiviral response. Therefore, initial sensing of rAdV or viral DNA (vDNA) does not depend on viral template transcription, but ML60218 treatment influences cellular cascades required for an antiviral response to rAdV. Using overexpression or knockdown assays, we examined how four DNA sensors influence the antiviral response. Knockdown of DNA Activator of Interferon (DAI) and p204, the murine ortholog to IFI16, had minimal influence on IRF3 phosphorylation. However, knockdown of absent in melanoma 2 (AIM2) and the helicase DDX41 resulted in diminished levels of (pser388)IRF3 following rAdV infection. Based on these data, multiple DNA sensors contribute to an antiviral DNA recognition response, leading to TBK1-dependent IRF3 phosphorylation in RAW 264.7 cells.
authors.affiliation: Weill Medical College of Cornell University, Department of Microbiology and Immunology, Molecular Biology Graduate Program, New York, New York, USA.
authors.forename: Saskia C
authors.initials: SC
authors.lastname: Stein
authors.forename: Erik
authors.initials: E
authors.lastname: Falck-Pedersen
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000260:Adenoviruses, Human / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D053487:DEAD-box RNA Helicases; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020868:Gene Silencing; D016679:Genome, Viral; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism*; D008264:Macrophages / Q000276:immunology* / Q000821:virology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012320:RNA Polymerase III / Q000037:antagonists & inhibitors; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic; D000099197:STING Protein
chemical_list: D053487:DEAD-box RNA Helicases; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D050838:Interferon Regulatory Factor-3; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D012320:RNA Polymerase III; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C540422:Aim2 protein, mouse; C489664:Ifi16 protein, mouse; C530855:Sting1 protein, mouse; D000099197:STING Protein; C402632:Zbp1 protein, mouse; C498588:Tbk1 protein, mouse; C567960:DDX41 protein, mouse