IFN regulatory factor family members differentially regulate the expression of type III IFN (IFN-lambda) genes

Virus replication induces the expression of antiviral type I (IFN-alphabeta) and type III (IFN-lambda1-3 or IL-28A/B and IL-29) IFN genes via TLR-dependent and -independent pathways. Although type III IFNs differ genetically from type I IFNs, their similar biological antiviral functions suggest that their expression is regulated in a similar fashion. Structural and functional characterization of the IFN-lambda1 and IFN-lambda3 gene promoters revealed them to be similar to IFN-beta and IFN-alpha genes, respectively. Both of these promoters had functional IFN-stimulated response element and NF-kappaB binding sites. The binding of IFN regulatory factors (IRF) to type III IFN promoter IFN-stimulated response element sites was the most important event regulating the expression of these genes. Ectopic expression of the components of TLR7 (MyD88 plus IRF1/IRF7), TLR3 (Toll/IL-1R domain-containing adapter-inducing factor), or retinoic acid-inducible gene I (RIG-I) signal transduction pathways induced the activation of IFN-lambda1 promoter, whereas the IFN-lambda3 promoter was efficiently activated only by overexpression of MyD88 and IRF7. The ectopic expression of Pin1, a recently identified suppressor for IRF3-dependent antiviral response, decreased the IFN promoter activation induced by any of these three signal transduction pathways, including the MyD88-dependent one. To conclude, the data suggest that the IFN-lambda1 gene is regulated by virus-activated IRF3 and IRF7, thus resembling that of the IFN-beta gene, whereas IFN-lambda2/3 gene expression is mainly controlled by IRF7, thus resembling those of IFN-alpha genes.     

Fas-associated death domain-containing protein-mediated antiviral innate immune signaling involves the regulation of Irf7

The induction of type I (alphabeta) IFN following virus infection is necessary for the stimulation of effective antiviral host defense. In fibroblasts, a subset of primary genes (including those encoding IFN-beta and IFN-alpha4) are induced directly by intracellular dsRNA generated by the virus during its replication. These primary type I IFNs induce expression of IFN regulatory factor (IRF)-7, required for production of a second cascade of IFN-alpha subtypes and the further establishment of a complete antiviral state. Previously, we had reported on a role for Fas-associated death domain-containing protein (FADD) in the control of TLR-independent innate immune responses to virus infection. Our data in this study demonstrate that FADD is not only required for efficient primary gene induction, but is also essential for induction of Irf7 and effective expression of secondary IFN-alphas and other antiviral genes. Ectopic overexpression of IRF-7 partially rescued dsRNA responsiveness and IFN-alpha production, and a constitutively active variant of IRF-7 displayed normal activity in Fadd(-/-) murine embryonic fibroblasts. MC159, a FADD-interacting viral protein encoded by the molluscum contagiosum poxvirus was found to inhibit dsRNA-activated signaling events upstream of IRF-7. These data indicate that FADD's antiviral activity involves regulation of IRF-7-dependent production of IFN-alpha subtypes and consequent induction of secondary antiviral genes.     

Critical role of a common transcription factor, IRF-1, in the regulation of IFN-beta and IFN-inducible genes.

Interferon regulatory factor 1 (IRF-1) is a protein that binds to cis-elements within the promoter of interferon (IFN)-beta and some IFN-inducible genes. We used a human fibroblast line, GM-637, to generate stable transfectants constitutively expressing IRF-1 mRNA in either the sense or antisense orientation. Upon induction with poly-(I).poly(C) or Newcastle disease virus, cells expressing sense IRF-1 mRNA produced significantly higher levels of IFN-beta mRNA and protein than control cells, whereas cells expressing antisense IRF-1 mRNA produced little or no IFN-beta mRNA and protein. Furthermore, clear differences were seen among the transfectants in the level of expression of two IFN-induced genes (2'-5'-oligoadenylate synthetase and class I HLA). Our data show that IRF-1 is essential for the induced expression of the IFN-beta gene. The results also indicate an important role of IRF-1 in the expression of IFN-inducible genes and suggest a role for IRF-1 in many other cytokine actions.     

Central role of interferon regulatory factor-1 (IRF-1) in controlling retinoic acid inducible gene-I (RIG-I) expression

Retinoic acid inducible gene-I (RIG-I) functions as the first line of defense against viral infection by sensing dsRNA and inducing type I interferon (IFN) production. The expression of RIG-I itself is induced by IFN-alpha/beta and dsRNA. To comprehend the molecular mechanism of expression regulation, we cloned the RIG-I promoter and analyzed its activity upon IFN-beta and dsRNA treatment. Under basal condition, RIG-I mRNA level and promoter activity were significantly higher in normal cells versus their tumor counterparts. In both normal and cancer cells, RIG-I expression was induced by IFN-beta and dsRNA. A single IRF-1 binding site in the proximal promoter functioned as a crucial regulator of basal, IFN-beta- and dsRNA-mediated induction of the RIG-I promoter. IFN-beta and dsRNA treatment increased IRF-1 binding to the RIG-I promoter. IRF-1 expression was also higher in normal cells than in cancer cells and it was induced by IFN-beta with similar kinetics as RIG-I. These results confirm that by controlling RIG-I expression, IRF-1 plays an essential role in anti-viral immunity. IRF-1 is a tumor suppressor and the expression profile of RIG-I together with its regulation by IRF-1 and the presence of a caspase-recruitment domain in RIG-I suggest that RIG-I might also possess tumor suppressor properties.     

Identification and partial characterization of FRAG-6, a novel interferon-stimulated gene that is expressed in an IRF-1-INDEPENDENT manner.

In order to identify new interferon-stimulated genes that could help in the better understanding of the mechanism of action of interferons (IFNs), we decided to compare, by differential display RT-PCR (DDRT-PCR), the pattern of gene expression between IFN-alpha treated and untreated mouse embryonic fibroblasts (MEFs). Here we describe the initial characterization of a new cDNA fragment, named FRAG-6, that is expressed only upon IFN stimulation. The IFN-induced expression of this new gene can be observed in both wild-type and IRF-1-deficient MEF. FRAG-6 cDNA hybridizes with an mRNA of 6-9 kb that is induced by IFNs in a time-dependent manner. Analysis of the cloned nucleotide sequence revealed a 174 amino acid (aa) open reading frame (ORF) contained within the 576 bp. No significant homology with known nucleotide or protein sequences was observed. FRAG-6 is induced in vitro upon treatment of wild type or IRF-1-null cells with IFN-alpha or -gamma, but not with TNF or IL-1. Treatment of mice with imiquimod, a potent inducer of IFN, led to induced expression of FRAG-6 mRNA in various organs from wild type or IRF-1-deficient mice, but not from STAT-1 or type I IFN receptor deficient animals. Our results demonstrate that FRAG-6 mRNA induction by interferons is IRF-1-independent and it is likely to be activated by the JAK/STAT pathway. Further characterization of FRAG-6 will help us in the understanding of the mechanism of action of IFNs.