Retinoic acid-inducible gene I mediates early antiviral response and Toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells.

Departments of Medicine, University of Texas Medical Branch, Galveston, Texas 77555-1060, USA.
Journal of Virology (Impact Factor: 4.65). 03/2007; 81(3):1401-11. DOI: 10.1128/JVI.01740-06
Source: PubMed

ABSTRACT Respiratory syncytial virus (RSV) is one of the most common viral pathogens causing severe lower respiratory tract infections in infants and young children. Infected host cells detect and respond to RNA viruses using different mechanisms in a cell-type-specific manner, including retinoic acid-inducible gene I (RIG-I)-dependent and Toll-like receptor (TLR)-dependent pathways. Because the relative contributions of these two pathways in the recognition of RSV infection are unknown, we examined their roles in this study. We found that RIG-I helicase binds RSV transcripts within 12 h of infection. Short interfering RNA (siRNA)-mediated RIG-I "knockdown" significantly inhibited early nuclear factor-kappaB (NF-kappaB) and interferon response factor 3 (IRF3) activation 9 h postinfection (p.i.). Consistent with this finding, RSV-induced beta interferon (IFN-beta), interferon-inducible protein 10 (IP-10), chemokine ligand 5 (CCL-5), and IFN-stimulated gene 15 (ISG15) expression levels were decreased in RIG-I-silenced cells during the early phase of infection but not at later times (18 h p.i.). In contrast, siRNA-mediated TLR3 knockdown did not affect RSV-induced NF-kappaB binding but did inhibit IFN-beta, IP-10, CCL-5, and ISG15 expression at late times of infection. Further studies revealed that TLR3 knockdown significantly reduced NF-kappaB/RelA transcription by its ability to block the activating phosphorylation of NF-kappaB/RelA at serine residue 276. We further found that TLR3 induction following RSV infection was regulated by RIG-I-dependent IFN-beta secreted from infected airway epithelial cells and was mediated by both IFN response-stimulated element (ISRE) and signal transducer and activator of transcription (STAT) sites in its proximal promoter. Together these findings indicate distinct temporal roles of RIG-I and TLR3 in mediating RSV-induced innate immune responses, which are coupled to distinct pathways controlling NF-kappaB activation.

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    ABSTRACT: RSV is a primary etiological agent of childhood lower respiratory tract disease. Molecular patterns induced by active infection trigger a coordinated retinoic acid-inducible gene (RIG)-I-toll like receptor (TLR) signaling response to induce inflammatory cytokines and anti-viral mucosal interferons. Recently we discovered a nuclear oxidative stress-sensitive pathway mediated by the DNA-damage response protein, ATM, in cytokine-induced NFκB/RelA Ser 276 phosphorylation. Here we, observe that ATM silencing results in enhanced ssRNA replication of RSV and Sendai Viruses, due to decreased expression and secretion of type -I and -III interferons (IFNs), despite maintenance of IRF3-dependent IFN-stimulated genes (ISGs). In addition to enhanced oxidative stress, RSV replication enhances foci of phosphorylated histone 2AX variant (γH2AX), Ser 1981 phosphorylation of ATM, and IKKγ/NEMO-dependent ATM nuclear export indicating activation of the DNA-damage response. ATM deficient cells show defective RSV-induced mitogen and stress-activated kinase (MSK)-1 Ser 376 phosphorylation and reduced RelA Ser 276 phosphorylation, whose formation is required for IRF7 expression. We observe that RelA binds inducibly binds the native IFN regulatory factor (IRF)-7 promoter in an ATM-dependent manner, and IRF7 inducibly binds to the endogenous retinoic acid inducible gene (RIG)-I promoter. Ectopic IRF7 expression restores RIG-I expression and type I/III IFN expression in ATM-silenced cells. We conclude that paramyxoviruses trigger the DNA-damage response, a pathway required for MSK1 activation of phospho Ser 276 RelA formation to trigger the IRF7-RIG-I amplification loop necessary for mucosal IFN production. These data provide molecular pathogenesis for defects in cellular innate immunity patients with homozygous ATM mutations. RNA virus infections trigger cellular response pathways to limit spread to adjacent tissues. This "innate immune response" is mediated by germline-encoded pattern recognition receptors that trigger activation of two, largely independent, intracellular NF-κB and IRF3 transcription factors. Downstream, expression of protective anti-viral interferons is amplified by positive feedback loops mediated by inducible interferon response factors (IRFs) and retinoic acid inducible gene (RIG-I). Our results indicate that a nuclear oxidative stress and DNA-damage sensing factor, ATM, is required to mediate a cross-talk pathway between NF-κB and IRF7 through mediating phosphorylation of NF-κB. Our studies provide further information about the defects in cellular- and innate immunity in patients with inherited ATM mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Journal of Virology 12/2014; 89(5). DOI:10.1128/JVI.02458-14 · 4.65 Impact Factor

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