IFN-stimulated Regulatory Element Research Articles

PK205R targets the proximal element of IFN-I signaling pathway to assist African swine fever virus to escape host innate immunity at the early stage of infection.

African swine fever virus (ASFV) is a nuclear cytoplasmic large DNA virus (NCLDV) that causes devastating hemorrhagic diseases in domestic pigs and wild boars, seriously threatening the development of the global pig industry. IFN-I plays an important role in the body's antiviral response. Similar to other DNA viruses, ASFV has evolved a variety of immune escape strategies to antagonize IFN-I signaling and maintain its proliferation. In this study, we showed that the ASFV early protein pK205R strongly inhibited interferon-stimulated genes (ISGs) as well as the promoter activity of IFN-stimulated regulatory elements (ISREs). Mechanistically, pK205R interacted with the intracellular domains of IFNAR1 and IFNAR2, thereby inhibiting the interaction of IFNAR1/2 with JAK1 and TYK2 and hindering the phosphorylation and nuclear translocation of STATs. Subsequently, we generated a recombinant strain of the ASFV-pK205R point mutation, ASFV-pK205R7PM. Notably, we detected higher levels of ISGs in porcine alveolar macrophages (PAMs) than in the parental strain during the early stages of ASFV-pK205R7PM infection. Moreover, ASFV-pK205R7PM attenuated the inhibitory effect on IFN-I signaling. In conclusion, we identified a new ASFV immunosuppressive protein that increases our understanding of ASFV immune escape mechanisms.

Identification of DreI as an Antiviral Factor Regulated by RLR Signaling Pathway

BackgroundRetinoic acid-inducible gene I (RIG-I)–like receptors (RLRs) had been demonstrated to prime interferon (IFN) response against viral infection via the conserved RLR signaling in fish, and a novel fish-specific gene, the grass carp reovirus (GCRV)-induced gene 2 (Gig2), had been suggested to play important role in host antiviral response.Methodology/Principal FindingsIn this study, we cloned and characterized zebrafish Gig2 homolog (named Danio rerio Gig2-I, DreI), and revealed its antiviral role and expressional regulation signaling pathway. RT-PCR, Western blot and promoter activity assay indicate that DreI can be induced by poly I:C, spring viremia of carp virus (SVCV) and recombinant IFN (rIFN), showing that DreI is a typical ISG. Using the pivotal signaling molecules of RLR pathway, including RIG-I, MDA5 and IRF3 from crucian carp, it is found that DreI expression is regulated by RLR cascade and IRF3 plays an important role in this regulation. Furthermore, promoter mutation assay confirms that the IFN-stimulated regulatory elements (ISRE) in the 5′ flanking region of DreI is essential for its induction. Finally, overexpression of DreI leads to establish a strong antiviral state against SVCV and Rana grylio virus (RGV) infection in EPC (Epithelioma papulosum cyprinid) cells.Conclusions/SignificanceThese data indicate that DreI is an antiviral protein, which is regulated by RLR signaling pathway.

MHC Class I Presentation and Regulation by IFN in Bony Fish Determined by Molecular Analysis of the Class I Locus in Grass Carp

Beyond their sequences, little is known regarding MHC class I presentation and regulation by IFN in bony fish. In this work, the class I locus (Ctid-UBA) was isolated from a grass carp fosmid library, and its polymorphisms and tissue expression were investigated. The Ctid-UBA and Ctid-beta2-microglobulin genes then were expressed and refolded, and tetramer techniques were used to identify the CTL response. The interaction between grass carp type I IFN and Ctid-UBA genes was investigated. Two fosmids coding for Ctid-UBA *0101 and Ctid-UBA *0201 genes were sequenced. The SXY box and IFN-stimulated regulatory element motifs were located from the start codons to -800 bp in Ctid-UBA. A Southern blot showed three to four bands, suggesting that grass carp contains at least three class I loci. In addition, the Ctid-UBA allelic genes are expressed in all tissue of grass carp. The three-dimensional structure of Ctid-UBA *0102 showed that the peptide-binding domain was formed by the alpha1 and alpha2 domains, which could bind several nonapeptides of grass carp hemorrhagic virus. There were 1.60% more PE-positive cells in P1(QPNEAIRSL)-immunized fish than in blank and adjuvant control groups. Additionally, recombinant grass carp IFN could regulate the expression of Ctid-UBA. These results characterize the class I presentation, CTL response, and regulation by type I IFN in bony fish.

Critical Role of the IFN-Stimulated Gene Factor 3 Complex in TLR-Mediated IL-27p28 Gene Expression Revealing a Two-Step Activation Process

In myeloid dendritic cells, activation of the IL-27p28 gene is selectively induced by ligands of TLR4 or TLR3, both coupled to the Toll/IL-1R-related domain-containing adaptor-inducing IFN/IFN regulatory factor (IRF)3 pathway. In response to both ligands, autocrine type 1 IFN production was required for optimal IL-27p28 expression. Type I IFN signaling was necessary for sustained IRF1 activation and formation of the IRF9-containing IFN-stimulated gene factor 3 complex. Indeed, we demonstrated that IRF1 and IRF9 are sequentially activated and recruited to the IL-27p28 IFN-stimulated regulatory element site. Involvement of IRF1 and IRF9 in the induction of IL-27p28 was confirmed in vitro and upon in vivo exposure to TLR ligands. Thus, in response to TLR4 or TLR3 ligation, the initial induction of the IL-27p28 gene depends on the recruitment of IRF1 and IRF3, whereas transcriptional amplification requires recruitment of the IFN-stimulated gene factor 3 complex. These results highlight the complex molecular interplay between TLRs and type I IFNs for the control of IL-27 synthesis.

Roles of IKK-β, IRF1, and p65 in the Activation of Chemokine Genes by Interferon-γ

Activation of chemokine genes in response to interferon (IFN)-gamma or NF-kappaB is an important aspect of inflammation. Using the chemokine gene ip-10 in mouse embryonic fibroblast cells as an example, we show that the response to IFN-gamma is long lasting but secondary: initial STAT1 activation drives IRF1 synthesis, and IRF1 then binds to IFN-stimulated regulatory elements (ISREs) in the ip-10 promoter. The promoters of most IKK-beta-dependent IFN-stimulated genes (ISGs) also contain ISREs. In response to IFN-gamma, inhibitor of NF-kappaB (IkappaB) kinase beta (IKK-beta) is required to activate both newly synthesized IRF1 and the p65 subunit of NF-kappaB, which contributes to ip-10 expression by binding to kappaB sites in the ip-10 promoter, with little or no activation of classical NF-kappaB. In contrast to IFN-gamma, IL-1beta induces ip-10 expression rapidly but transiently, by activating classical NF-kappaB and increasing the synthesis of IRF1. Together, IL-1beta and IFN-gamma induce ip-10 synergistically. IFN-gamma does not affect the transient activation of classical NF-kappaB by IL-1beta and synergistic induction of ip-10 expression by IFN-gamma and IL-1beta occurs even after the activation of classical NF-kappaB has returned to basal levels. Therefore, IKK-beta has a novel role in IFN-gamma-dependent activation of chemokine gene expression through its activation of IRF1 and p65.

Interferon-α-induced mTOR activation is an anti-hepatitis C virus signal via the phosphatidylinositol 3-kinase-Akt-independent pathway

The interferon-induced Jak-STAT signal alone is not sufficient to explain all the biological effects of IFN. The PI3-K pathways have emerged as a critical additional component of IFN-induced signaling. This study attempted to clarify that relationship between IFN-induced PI3-K-Akt-mTOR activity and anti-viral action. When the human normal hepatocyte derived cell line was treated with rapamycin (rapa) before accretion of IFN-alpha, tyrosine phosphorylation of STAT-1 was diminished. Pretreatment of rapa had an inhibitory effect on the IFN-alpha-induced expression of PKR and p48 in a dose dependent manner. Rapa inhibited the IFN-alpha inducible IFN-stimulated regulatory element luciferase activity in a dose-dependent manner. However, wortmannin, LY294002 and Akt inhibitor did not influence IFN-alpha inducible luciferase activity. To examine the effect of PI3-K-Akt-mTOR on the anti-HCV action of IFN-alpha, the full-length HCV replication system, OR6 cells were used. The pretreatment of rapa attenuated its anti-HCV replication effect in comparison to IFN-alpha alone, whereas the pretreatment with PI3-K inhibitors, wortmannin and LY294002 and Akt inhibitor did not influence IFN-induced anti-HCV replication. IFN-induced mTOR activity, independent of PI3K and Akt, is the critical factor for its anti-HCV activity. Jak independent mTOR activity involved STAT-1 phosphorylation and nuclear location, and then PKR is expressed in hepatocytes.

Human T-cell leukemia virus type 1 Tax modulates interferon-α signal transduction through competitive usage of the coactivator CBP/p300

We describe here Tax protein of human T-cell leukemia virus type 1 (HTLV-1) as an interferon (IFN)-α antagonist counteracting the transactivation function of IFN-stimulated gene factor 3 (ISGF3). Co-expression of Tax, but not the Tax mutant unable to bind to CBP, significantly inhibited the reporter gene expression directed by IFN-stimulated regulatory elements, despite that the formation of DNA-binding ISGF3 complex was unaffected. Gene activation induced by STAT2 transcription domain was also inhibited by expression of Tax. Furthermore, Tax-mediated transcriptional inhibition was reversed by overexpression of p300. These observations indicate that Tax interferes with IFN-α-induced JAK-STAT pathway by competition with STAT2 for CBP/p300 binding. Consistently, GST pull-down assay showed that Tax dose-dependently inhibited binding of STAT2 to p300. This study suggests that Tax may prevent IFN-α from exerting its antiviral, antiproliferative and proapoptotic effects, thereby contributing to persistent viral infection and HTLV-1-associated oncogenesis.

Differential effects of calcineurin inhibitors, tacrolimus and cyclosporin a, on interferon-induced antiviral protein in human hepatocyte cells

The premise of our study is that selective inhibition of interferon (IFN) by calcineurin inhibitors contribute to the increased severity of hepatitis C virus (HCV) posttransplantation. Therefore, we examined the influence of calcineurin inhibitors in the human hepatocyte cell line on IFN-alpha-induced phosphorylation of Janus kinase (Jak) and signal transducers and activators of transcription (STAT), nuclear translocation of IFN-stimulated gene factor 3 (ISGF-3), IFN-stimulated regulatory element (ISRE)-contained promoter activity, and the expressions of antiviral proteins. Tacrolimus (Tac), but not cyclosporin A (CyA), had an inhibitory effect on IFN-alpha-induced double-stranded ribonucleic acid (RNA)-dependent protein kinase (PKR) in a dose-dependent manner. STAT-1 also acted in a similar fashion to PKR. IFN-alpha combined with Tac attenuated the ISRE-containing promoter gene activity as compared with IFN-alpha alone. In contrast, its expression in pretreated CyA was slightly attenuated. In pretreated Tac, but not CyA, the levels of IFN-alpha-induced tyrosine phosphorylated STAT-1 and -2 were clearly lower than those induced by IFN-alpha alone. Tac and CyA did not decrease the IFN-alpha-induced JAK-1 phosphorylation. The nuclear translocation rate of tyrosine phosphorylated STAT-1 was inhibited by pretreatment of both Tac and CyA by western blotting and immunohistochemistry. In an HCV replicon system, pretreated Tac diminished the replication inhibitory effect of IFN-alpha. In this study, we show that calcineurin inhibitors, especially Tac, are the negative regulators of IFN signaling in the hepatocyte; the greatest cause of such inhibition is the phosphorylation disturbance of STAT-1, next to inhibition of the nuclear translocation of STAT-1. In conclusion, disturbance of tyrosine phosphorylation of STAT-1 resulted in diminished ISRE-containing promoter activity and a decline in antiviral protein expression. Moreover, the replication of HCV was activated. This phenomenon is detrimental to IFN therapy after liver transplantation, and the selection of calcineurin inhibitors may warrant further discussion depending on the transplant situation.

Basic aspects of the treatment for hepatitis C: mechanisms of action of interferon alpha and ribavirin and the bases of individualization

The treatment of patients with chronic hepatitis C hasdeveloped considerably in recent years. However, it is stillbased on the use of interferon alpha (IFN-α) as an antiviraland immunomodulatory agent against the hepatitis C virus(HCV).The IFNs are a family of proteins that are naturallyproduced by the cells of the immune system. The IFN-a proteinpresents antiviral, antiproliferative and immunomodulatoryactivity [1-3]. Its mechanism of biological action occursthrough the activation of specific genes, influencing cellgrowth and division, as well as modulating some immunesystem activities. Therefore, IFNs have an indirect antiviraleffect on HCV [2,4].Commercially, IFN- α is produced by means of recombinantDNA techniques and is available in preparations of two distinctsubtypes (IFN-α 2a or IFN-α 2b), which can be combinedwith other molecules, such as polyethylene glycolor, morerecently, albumin [5,6]. The only difference between IFN- α 2aand IFN- α 2b is in the amino acid present at position 23 of theprotein: IFN- α 2a has a lysine at that position, whereas IFN- α2b has an arginine [7].After the binding with its specific receptor (IFNAR) onthe surface of the target cells, IFN- α activates an intracellularsignaling cascade, which takes the induction of IFN-stimulatedgenes (ISGs), establishing a non-virus-specific antiviral stateinside the cell [3,7]. The principal signaling mechanism usedby IFN- α is the so-called Janus kinase/signal transducers andactivators of transcription (Jak/STAT) pathway [3]. Therefore,two cytoplasmatic proteins with the activity of tyrosine kinaseassociated with IFNAR, activated Jak1 and tyrosine kinase 2(Tyk2), are activated by the dimerization of the receptors.Activated Jak1 and Tyk2 perform the phosphorylation ofSTAT1 and STAT2, respectively. The phosphorylated STAT1and STAT2 bond with the protein p48 forming IFN-stimulatedgene factor 3 (ISGF3), which translocates into the nucleusand bonds with IFN-stimulated regulatory element in thesequences which promote a variety of genes inducible byIFN-α including antiviral proteins such as 2’5’-oligoadenylatesynthetase (2’5’OAS), protein kinase RNA, and Mx protein[1,3,7,8].The absorption of IFN-α (2a or 2b) is high (above 80%)when administrated intramuscularly or subcutaneously. Theconcentration typically peaks at 3-12 h after administration [9].The metabolism and elimination of IFN- α occurs principallyvia the kidneys, with a half-life of 3-8 h [9].

The RNA Helicase Lgp2 Inhibits TLR-Independent Sensing of Viral Replication by Retinoic Acid-Inducible Gene-I

The paramyxovirus Sendai (SV), is a well-established inducer of IFN-alphabeta gene expression. In this study we show that SV induces IFN-alphabeta gene expression normally in cells from mice with targeted deletions of the Toll-IL-1 resistance domain containing adapters MyD88, Mal, Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF), and TRIF-related adaptor molecule TLR3, or the E3 ubiquitin ligase, TNFR-associated factor 6. This TLR-independent induction of IFN-alphabeta after SV infection is replication dependent and mediated by the RNA helicase, retinoic acid-inducible gene-I (RIG-I) and not the related family member, melanoma differentiation-associated gene 5. Furthermore, we characterize a RIG-I-like RNA helicase, Lgp2. In contrast to RIG-I or melanoma differentiation-associated gene 5, Lgp2 lacks signaling caspase recruitment and activation domains. Overexpression of Lgp2 inhibits SV and Newcastle disease virus signaling to IFN-stimulated regulatory element- and NF-kappaB-dependent pathways. Importantly, Lgp2 does not prevent TLR3 signaling. Like RIG-I, Lgp2 binds double-stranded, but not single-stranded, RNA. Quantitative PCR analysis demonstrates that Lgp2 is present in unstimulated cells at a lower level than RIG-I, although both helicases are induced to similar levels after virus infection. We propose that Lgp2 acts as a negative feedback regulator of antiviral signaling by sequestering dsRNA from RIG-I.

Early Growth Response-1 Regulates Lipopolysaccharide-induced Suppressor of Cytokine Signaling-1 Transcription

Suppressor of cytokine signaling (SOCS)-1 is a critical regulator of lipopolysaccharide (LPS) tolerance and LPS-induced cytokine production. The mechanisms regulating the transcription of SOCS-1 in response to LPS are not entirely understood. Functional analysis of the SOCS-1 promoter demonstrates that early growth response-1 (Egr-1) is an important transcriptional regulator of SOCS-1. Two Egr-1 binding sites are present within the SOCS-1 promoter as shown by EMSA and supershift analysis. Further, mutation of the Egr-1 binding sites significantly reduces both the basal and LPS-induced transcriptional activity of the promoter. Chromatin immunoprecipitation experiments confirm LPS-induced binding of Egr-1 to the SOCS-1 promoter in vivo. Additionally, Egr-1(-/-) macrophages show reduced levels of LPS-induced SOCS-1 expression in comparison with macrophages derived from Egr-1(+/+) littermate controls. These results demonstrate an important role for Egr-1 in regulating both the basal and LPS-induced activity of the SOCS-1 promoter.

Involvement of IL-1β and IL-10 in IFN-α-mediated antiviral gene induction in human hepatoma cells

Crosstalk between interferons (IFNs) and several cytokines is likely to play an important role in viral clearance in chronic hepatitides B and C. We investigated the influence of this phenomenon on IFN-inducible antiviral gene expression in HuH-7 human hepatoma cells. HuH-7 cells were treated with IFN-α in the absence or presence of interleukin-1β (IL-1β) or IL-10 and the expression of antiviral genes such as 2 ′5 ′-oligoadenylate synthetase (2 ′5 ′-OAS) and double-stranded RNA-dependent protein kinase (PKR), as well as activation of signal transducer and activator of transcription 1 (STAT1), a key step for relaying the IFN-α signals, was analyzed by Northern blotting, Western blotting, and the reporter gene transfection assay. IL-1β potentiated IFN-α-induced 2 ′5 ′-OAS and PKR gene expression, similar to expression of the transfected reporter genes containing the IFN-stimulated regulatory elements, while IL-10 suppressed IFN-α-stimulated gene expression. With regard to IFN-α signaling, IL-1β enhanced both tyrosine and serine phosphorylation of STAT1 through p38 mitogen-activated protein kinase activation. In contrast, IL-10 inhibited IFN-α-mediated tyrosine phosphorylation of STAT1 by induction of a Janus kinase inhibitor, JAB. IL-1β and IL-10 interact with IFN-α to up- and down-regulate antiviral gene expression, respectively, by modulating STAT1 activation induced by IFN-α.

Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes.

Bacterial lipopolysaccharide (LPS) triggers innate immune responses through Toll-like receptor (TLR) 4, a member of the TLR family that participates in pathogen recognition. TLRs recruit a cytoplasmic protein, MyD88, upon pathogen recognition, mediating its function for immune responses. Two major pathways for LPS have been suggested in recent studies, which are referred to as MyD88-dependent and -independent pathways. We report in this study the characterization of the MyD88-independent pathway via TLR4. MyD88-deficient cells failed to produce inflammatory cytokines in response to LPS, whereas they responded to LPS by activating IFN-regulatory factor 3 as well as inducing the genes containing IFN-stimulated regulatory elements such as IP-10. In contrast, a lipopeptide that activates TLR2 had no ability to activate IFN-regulatory factor 3. The MyD88-independent pathway was also activated in cells lacking both MyD88 and TNFR-associated factor 6. Thus, TLR4 signaling is composed of at least two distinct pathways, a MyD88-dependent pathway that is critical to the induction of inflammatory cytokines and a MyD88/TNFR-associated factor 6-independent pathway that regulates induction of IP-10.

Synergistic Induction of the Tap-1 Gene by IFN-γ and Lipopolysaccharide in Macrophages Is Regulated by STAT1

Proper regulation of the Tap-1 gene is critical for the initiation and continuation of a cellular immune response. Analysis of the Tap-1/low molecular mass polypeptide 2 bidirectional promoter showed that the IFN-gamma activation site element is critical for the rapid induction of the promoter by IFN-gamma following transfection into the human macrophage cell line THP-1. Furthermore, activation of STAT1 binding to this site was important for the synergistic response seen following the stimulation with both IFN-gamma and LPS. Mutation of an IFN-stimulated regulatory element that binds IFN regulatory factor 1 appeared to enhance the response to IFN-gamma and LPS. These data show that STAT1 is necessary for the activation of Tap-1 gene expression in APCs and initiation of cellular immune responses. Furthermore, our data suggest that bacterial products such as LPS may enhance cellular immune responses through augmenting the ability of STAT1 to regulate IFN-gamma-inducible genes.

Deficiency in the transcription factor interferon regulatory factor (IRF)-2 leads to severely compromised development of natural killer and T helper type 1 cells.

Interferon (IFN) regulatory factor (IRF)-2 was originally described as an antagonist of IRF-1-mediated transcriptional regulation of IFN-inducible genes. IRF-1(-/)- mice exhibit defective T helper type 1 (Th1) cell differentiation. We have used experimental leishmaniasis to show that, like IRF-1(-/)- mice, IRF-2(-/)- mice are susceptible to Leishmania major infection due to a defect in Th1 differentiation. Natural killer (NK) cell development is compromised in both IRF-1(-/)- and IRF-2(-/)- mice, but the underlying mechanism differs. NK (but not NK(+) T) cell numbers are decreased in IRF-2(-/)- mice, and the NK cells that are present are immature in phenotype. Therefore, like IRF-1, IRF-2 is required for normal generation of Th1 responses and for NK cell development in vivo. In this particular circumstance the absence of IRF-2 cannot be compensated for by the presence of IRF-1 alone. Mechanistically, IRF-2 may act as a functional agonist rather than antagonist of IRF-1 for some, but not all, IFN-stimulated regulatory element (ISRE)-responsive genes.

Evidence for glycosphingolipid modification of the type 1 IFN receptor.

P1-determinant glycolipids that include two membrane glycosphingolipids, globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc-ceramide, Gb3) and galabiosylceramide (Gal alpha 1-4Gal-ceramide, Gb2) are receptors for an Escherichia coli-derived subunit toxin, verotoxin (VT-1). Studies with Daudi cells and glycosphingolipid-deficient Daudi mutants and U937 cells identified that the presence of Gb2/Gb3 correlates with IFN-alpha sensitivity. Comparison of amino acid sequences between VT-1 and the extracellular region of the 63-kDa IFN-alpha beta receptor (IFNAR) peptide reveals regions of identity, specifically in those domains in the VT-1 B subunit, that have been implicated as Gb2/Gb3 binding sites. In direct ligand binding studies, we show that membrane Gb2/Gb3 content affects the binding capacity of cells for IFN-alpha, although IFNAR cell surface expression is unaffected. Binding of IFN-alpha to the receptor leads to kinase-associated phosphorylation of the latent transcription factor, ISGF3, which activates transcription by binding to IFN-stimulated regulatory elements in IFN-sensitive genes. Electrophoretic mobility band shift assays indicated that U-937 and Daudi mutant cells, deficient in Gb2 and Gb3, exhibited reduced nuclear factor binding to the human 2-5A synthetase IFN-stimulated regulatory element when compared with wild-type Daudi cells, after exposure to IFN-alpha. Moreover, when Daudi cells were treated with a ceramide analogue, 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol, Gb2 and Gb3 synthesis was inhibited and a concomitant reduction in IFN-induced ISGF3 activation was noted. IFNAR cell surface expression was unaffected by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol treatment. By using a fusion protein of the extracellular domain of IFNAR linked at the carboxyl terminus to the Fc portion of IgG1, we demonstrate that IFNAR is able to bind preferentially to Gb2. These results suggest that an association of IFNAR with membrane Gal alpha 1-4Gal containing glycolipids facilitates receptor-mediated signaling.

Rapid activation by interferon alpha of a latent DNA-binding protein present in the cytoplasm of untreated cells.

The highly conserved interferon (IFN)-stimulated regulatory elements of the human genes 6-16 and 9-27 bind to one or more proteins (E factor) detected in extracts of human Bristol 8 B cells or human foreskin fibroblast cells treated with IFN-alpha. E factor is not detectable in extracts of untreated cells and appears in IFN-treated cells within less than 1 min in a form extractable with low salt and thus presumably not bound to DNA. After a few more minutes, the level of this form decreases in parallel with the increase of a form extractable only with high salt and thus presumably bound to DNA. Induction of E factor by IFN-alpha can occur in nuclei-free cytoplasts, whereas no E factor was detected in IFN-treated nucleoplasts. Together, these results suggest a model for signal transduction in which latent E factor, located in the cytoplasm, is activated or released from an inhibitor very rapidly upon binding of IFN-alpha to its receptor. Active E factor can then migrate to the nucleus, where it binds to the IFN-stimulated regulatory elements of IFN-regulated genes, activating their transcription.