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1.  Cytokines Induce an Early Steroid Resistance in Airway Smooth Muscle Cells 
We have previously shown that long-term treatment of airway smooth muscle (ASM) cells with a combination of TNF-α and IFN-γ impaired steroid anti-inflammatory action through the up-regulation of glucocorticoid receptor beta isoform (GRβ) (Mol Pharmacol 2006;69:588–596). We here found that steroid actions could also be suppressed by short-term exposure of ASM cells to TNF-α and IFN-γ (6 h) as shown by the abrogated glucocorticoid responsive element (GRE)-dependent gene transcription; surprisingly, neither GRα nuclear translocation nor GRβ expression was affected by cytokine mixture. The earlier induction of CD38, a molecule recently involved in asthma, seen with TNF-α and IFN-γ combination but not with cytokine alone, was also completely insensitive to steroid pretreatment. Chromatin-immunoprecipitation (IP) and siRNA strategies revealed not only increased binding of interferon regulatory factor 1 (IRF-1) transcription factor to CD38 promoter, but also its implication in regulating CD38 gene transcription. Interestingly, the capacity of fluticasone to completely inhibit TNF-α–induced IRF-1 expression, IRF-1 DNA binding, and transactivation activities was completely lost in cells exposed to TNF-α and IFN-γ in combination. This early steroid dysfunction seen with cytokine combination could be reproduced by enhancing IRF-1 cellular levels using constitutively active IRF-1, which dose-dependently inhibited GRE-dependent gene transcription. Consistently, reducing IRF-1 cellular levels using siRNA approach significantly restored steroid transactivation activities. Collectively, our findings demonstrate for the first time that IRF-1 is a novel alternative GRβ-independent mechanism mediating steroid dysfunction induced by pro-asthmatic cytokines, in part via the suppression of GRα activities.
doi:10.1165/rcmb.2007-0226OC
PMCID: PMC2274949  PMID: 17947510
transcription factor; glucocorticoid; inflammation; asthma; mesenchymal cells
2.  Functional Analysis of Human Herpesvirus 8-Encoded Viral Interferon Regulatory Factor 1 and Its Association with Cellular Interferon Regulatory Factors and p300 
Journal of Virology  1999;73(9):7334-7342.
Human herpesvirus 8/Kaposi sarcoma-associated virus (HHV-8/KSHV) contains, in addition to genes required for viral replication, a unique set of nonstructural genes which may be part of viral mimicry and contribute to viral replication and pathogenesis in vivo. Among these, HHV-8 encodes four open reading frames (ORFs) that showed homology to the transcription factors of the interferon regulatory factor (IRF) family. The ORF K9, viral IRF 1 (vIRF-1), has been cloned, and it was shown that, when overexpressed, it down modulates the interferon-mediated transcriptional activation of the interferon-stimulated gene 15 (ISG 15) promoter, and the role of vIRF-1 in viral mimicry was implied. However, the molecular mechanism of this effect has not been clarified. Here, we extend this observation and show that vIRF-1 also downregulates the transcriptional activity of IFNA gene promoter in infected cells by interfering with the transactivating activity of cellular IRFs, including IRF-1 and IRF-3. We further show that ectopic expression of vIRF-1 in NIH 3T3 cells confers resistance to tumor necrosis factor alpha-induced apoptosis. While vIRF-1 is unable to bind DNA with the same specificity as cellular IRFs, we demonstrate by in vitro binding assay that it can associate with the family of cellular IRFs, such as IRF-1 and the interferon consensus sequence binding protein. vIRF-1 interaction domain was localized between amino acids (aa) 152 and 243. While no binding between the full-size IRF-3 and vIRF-1 could be detected by the same assay, we show that vIRF-1 also targets the carboxy-terminal region (aa 1623 to 2414) of the transcriptional coactivator p300 which could also bind IRF-3 and IRF-1. These results demonstrate that vIRF-1 can modulate the transcription of the IFNA genes by direct heterodimerization with members of the IRF family, as well as by competitive binding with cellular transcription factors to the carboxy-terminal region of p300.
PMCID: PMC104259  PMID: 10438822
3.  Bim Nuclear Translocation and Inactivation by Viral Interferon Regulatory Factor 
PLoS Pathogens  2010;6(8):e1001031.
Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of the host cell. Human herpesvirus 8 (HHV-8) uses several strategies to block the host's innate antiviral defenses via interference with interferon and apoptotic signaling. Contributors include the four viral interferon regulatory factors (vIRFs 1–4), which function in dominant negative fashion to block cellular IRF activities in addition to targeting IRF signaling-induced proteins such as p53 and inhibiting other inducers of apoptosis such as TGFβ receptor-activated Smad transcription factors. Here we identify direct targeting by vIRF-1 of BH3-only pro-apoptotic Bcl-2 family member Bim, a key negative regulator of HHV-8 replication, to effect its inactivation via nuclear translocation. vIRF-1-mediated relocalization of Bim was identified in transfected cells, by both immunofluorescence assay and western analysis of fractionated cell extracts. Also, co-localization of vIRF-1 and Bim was detected in nuclei of lytically infected endothelial cells. In vitro co-precipitation assays using purified vIRF-1 and Bim revealed direct interaction between the proteins, and Bim-binding residues of vIRF-1 were mapped by deletion and point mutagenesis. Generation and experimental utilization of Bim-refractory vIRF-1 variants revealed the importance of vIRF-1:Bim interaction, specifically, in pro-replication and anti-apoptotic activity of vIRF-1. Furthermore, blocking of the interaction with cell-permeable peptide corresponding to the Bim-binding region of vIRF-1 confirmed the relevance of vIRF-1:Bim association to vIRF-1 pro-replication activity. To our knowledge, this is the first report of an IRF protein that interacts with a Bcl-2 family member and of nuclear sequestration of Bim or any other member of the family as a means of inactivation. The data presented reveal a novel mechanism utilized by a virus to control replication-induced apoptosis and suggest that inhibitory targeting of vIRF-1:Bim interaction may provide an effective antiviral strategy.
Author Summary
Human herpesvirus 8 (HHV-8) is a pathogen associated with cancers Kaposi's sarcoma (KS), an endothelial cell disease, and B cell malignancies primary effusion lymphoma and multicentric Castleman's disease. KS is particularly prevalent amongst HIV-positive populations in Africa and is a major health concern. Virus productive replication, in addition to latency, is important for maintaining viral load within the host and also for KS pathogenesis. Essential to HHV-8 and other virus replication is the control of innate host defenses, which comprise stress-sensing cellular signaling pathways that result ultimately in programmed cell death (apoptosis). Here we identify a novel mechanism whereby a viral protein, viral interferon regulatory factor-1 (vIRF-1), mediates inhibition of a stress sensor and initiator of apoptosis, Bim, by inducing its translocation to the cell nucleus and thereby sequestration away from the cytoplasmic compartment where it exerts its pro-death activity. We show that vIRF-1:Bim interaction is necessary for efficient HHV-8 productive replication and that it can be blocked using a cell-permeable antagonist of vIRF-1:Bim binding. Our data not only identify previously unsuspected mechanisms of Bim inactivation and vIRF-1 function, but suggest that inhibitory targeting of vIRF-1 interaction with Bim may be of therapeutic benefit.
doi:10.1371/journal.ppat.1001031
PMCID: PMC2916877  PMID: 20700448
4.  Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor confers resistance to the antiproliferative effect of interferon-alpha. 
Molecular Medicine  1998;4(6):402-412.
BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a 442 amino acid polypeptide-designated viral interferon regulatory factor (vIRF) that displays homology to members of the interferon regulatory factor (IRF) family that bind to consensus interferon sequences and transactivate cellular genes that can modulate growth inhibition. Studies were conducted to determine whether vIRF affects the growth suppression mediated by interferon-alpha (IFN-alpha) in a human B lymphocyte cell line. MATERIALS AND METHODS: The human B lymphocyte cell line Daudi, which is sensitive to the antiproliferative effects of IFN-alpha, was stably transfected to express vIRF, and the proliferative response of vIRF expressing cells to IFN-alpha was compared with controls. The effect of vIRF on IRF- 1 transactivation was analyzed by co-transfection of an IFN-alpha-responsive chloramphenicol acetyltransferase reporter and expression plasmids encoding IRF-1 and vIRF. Electrophoretic mobility shift assays were conducted to determine whether vIRF interferes with the DNA binding activity of IRF-1. RESULTS: Daudi human B lymphocyte cells expressing vIRF were resistant to the antiproliferative effects of IFN-alpha, whereas wild-type Daudi or Daudi cells transformed with vector DNA were growth inhibited by IFN-alpha. The activation of an interferon-responsive reporter by IFN-alpha or IRF-1 was repressed by expression of vIRF. IRF-1 DNA binding activity was unaffected by vIRF, and vIRF alone did not bind to the interferon consensus sequence. CONCLUSIONS: These studies revealed that vIRF functions to inhibit interferon-mediated growth control of a human B lymphocyte cell line by targeting IRF-1 transactivation of interferon-inducible genes. Since KSHV is a B lymphotropic herpesvirus associated with two forms of B lymphocyte neoplasms, these effects of vIRF likely contribute to B cell oncogenesis associated with KSHV infection.
Images
PMCID: PMC2230276  PMID: 10780883
5.  Loss of interferon regulatory factor 5 (IRF5) expression in human ductal carcinoma correlates with disease stage and contributes to metastasis 
Breast Cancer Research : BCR  2011;13(6):R111.
Introduction
New signaling pathways of the interleukin (IL) family, interferons (IFN) and interferon regulatory factors (IRF) have recently been found within tumor microenvironments and in metastatic sites. Some of these cytokines stimulate while others inhibit breast cancer proliferation and/or invasion. IRFs, a family of nine mammalian transcription factors, have multiple biologic functions that when dysregulated may contribute to tumorigenesis; most well-known are their roles in regulating/initiating host immunity. Some IRF family members have been implicated in tumorigenesis yet little is still known of their expression in primary human tumors or their role(s) in disease development/progression. IRF5 is one of the newer family members to be studied and has been shown to be a critical mediator of host immunity and the cellular response to DNA damage. Here, we examined the expression of IRF5 in primary breast tissue and determined how loss of expression may contribute to breast cancer development and/or progression.
Methods
Formalin-fixed paraffin-embedded archival breast tissue specimens from patients with atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) were examined for their expression of IRF1 and IRF5. Knockdown or overexpression of IRF5 in MCF-10A, MCF-7 and MDA-MB-231 mammary epithelial cell lines was used to examine the role of IRF5 in growth inhibition, invasion and tumorigenesis.
Results
Analysis of IRF expression in human breast tissues revealed the unique down-regulation of IRF5 in patients with different grades of DCIS and IDC as compared to IRF1; loss of IRF5 preceded that of IRF1 and correlated with increased invasiveness. Overexpression of IRF5 in breast cancer cells inhibited in vitro and in vivo cell growth and sensitized them to DNA damage. Complementary experiments with IRF5 siRNAs made normal mammary epithelial cells resistant to DNA damage. By 3-D culture, IRF5 overexpression reverted MDA-MB-231 to normal acini-like structures; cells overexpressing IRF5 had decreased CXCR4 expression and were insensitive to SDF-1/CXCL12-induced migration. These findings were confirmed by CXCR4 promoter reporter assays.
Conclusions
IRF5 is an important tumor suppressor that regulates multiple cellular processes involved in the conversion of normal mammary epithelial cells to tumor epithelial cells with metastatic potential.
doi:10.1186/bcr3053
PMCID: PMC3326553  PMID: 22053985
6.  Rotavirus NSP1 Mediates Degradation of Interferon Regulatory Factors through Targeting of the Dimerization Domain 
Journal of Virology  2013;87(17):9813-9821.
Rotavirus nonstructural protein NSP1 can inhibit expression of interferon (IFN) and IFN-stimulated gene products by inducing proteasome-mediated degradation of IFN-regulatory factors (IRFs), including IRF3, IRF5, and IRF7. All IRF proteins share an N-terminal DNA-binding domain (DBD), and IRF3, IRF5, and IRF7 contain a similar C-proximal IRF association domain (IAD) that mediates IRF dimerization. An autoinhibitory domain (ID) at the extreme C terminus interacts with the IAD, burying residues necessary for IRF dimerization. Phosphorylation of serine/threonine residues in the ID induces charge repulsions that unmask the IAD, enabling IRF dimerization and subsequent nuclear translocation. To define the region of IRF proteins targeted for degradation by NSP1, we generated IRF3 and IRF7 truncation mutants and transiently expressed each with simian SA11-4F NSP1. These assays indicated that the IAD represented a necessary and sufficient target for degradation. Because NSP1 did not mediate degradation of truncated forms of the IAD, NSP1 likely requires a structurally intact IAD for recognition and targeting of IRF proteins. IRF9, which contains an IAD-like region that directs interactions with signal inducer and activator of transcription (STAT) proteins, was also targeted for degradation by NSP1, while IRF1, which lacks an IAD, was not. Analysis of mutant forms of IRF3 unable to undergo dimerization or that were constitutively dimeric showed that both were targeted for degradation by NSP1. These results indicate that SA11-4F NSP1 can induce degradation of inactive and activated forms of IAD-containing IRF proteins (IRF3 to IRF9), allowing a multipronged attack on IFN-based pathways that promote antiviral innate and adaptive immune responses.
doi:10.1128/JVI.01146-13
PMCID: PMC3754143  PMID: 23824805
7.  Cleavage of Interferon Regulatory Factor 7 by Enterovirus 71 3C Suppresses Cellular Responses 
Journal of Virology  2013;87(3):1690-1698.
Enterovirus 71 (EV71) is a positive-stranded RNA virus which is capable of inhibiting innate immunity. Among virus-encoded proteins, the 3C protein compromises the type I interferon (IFN-I) response mediated by retinoid acid-inducible gene-I (RIG-I) or Toll-like receptor 3 that activates interferon regulatory 3 (IRF3) and IRF7. In the present study, we report that enterovirus 71 downregulates IRF7 through the 3C protein, which inhibits the function of IRF7. When expressed in mammalian cells, the 3C protein mediates cleavage of IRF7 rather than that of IRF3. This process is insensitive to inhibitors of caspase, proteasome, lysosome, and autophagy. H40D substitution in the 3C active site abolishes its activity, whereas R84Q or V154S substitution in the RNA binding motif has no effect. Furthermore, 3C-mediated cleavage occurs at the Q189-S190 junction within the constitutive activation domain of IRF7, resulting in two cleaved IRF7 fragments that are incapable of activating IFN expression. Ectopic expression of wild-type IRF7 limits EV71 replication. On the other hand, expression of the amino-terminal domain of IRF7 enhances EV71 infection, which correlates with its ability to interact with and inhibit IRF3. These results suggest that control of IRF7 by the 3C protein may represent a viral mechanism to escape cellular responses.
doi:10.1128/JVI.01855-12
PMCID: PMC3554134  PMID: 23175366
8.  TRIM28 is a SUMO E3 Ligase and Negative Regulator of Interferon Regulatory Factor 7 
Interferon regulatory factor 7 (IRF7) is a potent transcription factor of type I interferons (IFNs) and IFN stimulated genes (ISGs) and is known as the master regulator of type I IFN-dependent immune responses. Because excessive responses could harm the host, IRF7 itself is delicately regulated at the transcriptional, translational, and posttranslational levels. Modification of IRF7 by small ubiquitin-related modifiers (SUMOs) has been shown to regulate IFN expression and antiviral responses negatively, but the specific E3 ligase needed for IRF7 SUMOylation has remained unknown. As reported here, we have identified the tripartite motif–containing (TRIM) protein 28 (TRIM28) as a binding partner of IRF7. We have demonstrated that TRIM28 also interacts with the SUMO E2 enzyme and increases SUMOylation of IRF7 both in vivo and in vitro, suggesting it acts as a SUMO E3 ligase of IRF7. Unlike the common SUMO E3 ligase, protein inhibitor of activated STAT 1(PIAS1), the E3 activity of TRIM28 is specific to IRF7, because it has little effect on IRF7’s close relative IRF3. TRIM28 is therefore, so far as we know, the first IRF7-specific SUMO E3 reported. TRIM28-mediated SUMOylation of IRF7 is increased during viral infection, and SUMOylation of transcription factors usually results in transcriptional repression. Overexpression of TRIM28 therefore inhibits IRF7 transactivation activity, whereas knockdown of TRIM28 has the opposite effect and potentiates IFN production and antiviral responses. Collectively, our results suggest that TRIM28 is a specific SUMO E3 ligase and negative regulator of IRF7.
doi:10.4049/jimmunol.1101704
PMCID: PMC3197880  PMID: 21940674
9.  Latently Expressed Human Herpesvirus 8-Encoded Interferon Regulatory Factor 2 Inhibits Double-Stranded RNA-Activated Protein Kinase 
Journal of Virology  2001;75(5):2345-2352.
Human herpesvirus 8 (HHV-8; Kaposi's sarcoma herpesvirus) encodes four open reading frames with homology to cellular proteins of interferon regulatory factor (IRF) family. Three of them, viral IRF-1 (vIRF-1), vIRF-2, and vIRF-3, have been cloned and found, when overexpressed, to down-regulate the transcriptional activity of interferon type I gene promoters in infected cells by interfering with the transactivating activity of cellular IRFs. In this study, we have further characterized vIRF-2 and shown that it is a nuclear protein which is constitutively expressed in HHV-8-positive pleural effusion lymphoma cell lines. Nuclear localization of vIRF-2 was confirmed by in situ detection of ectopically expressed enhanced green fluorescent protein/vIRF-2 fusion protein. We found that the expression of vIRF-2 in HEK293 cells inhibited the antiviral effect of interferon and rescued translation of vesicular stomatitis virus mRNA from interferon-induced translational block. To provide insight into the mechanism of this effect we have demonstrated that vIRF-2 physically interacts with PKR consequently inhibiting autophosphorylation of double-stranded RNA-activated protein kinase (PKR) and blocking phosphorylation of PKR substrates histone 2A and eukaryotic translation initiation factor 2α. These results suggest that the latently expressed vIRF-2 has a role in viral mimicry which targets the activity of interferon-induced PKR kinase. By inhibiting the kinase activity of PKR and consequent down-modulation of protein synthesis, HHV-8 has evolved a mechanism by which it can overcome the interferon-mediated antiviral effect. Thus, the anti-interferon functions of vIRF-2 may contribute to the establishment of a chronic or latent infection.
doi:10.1128/JVI.75.5.2345-2352.2001
PMCID: PMC114818  PMID: 11160738
10.  Characterization of a Novel Human Herpesvirus 8-Encoded Protein, vIRF-3, That Shows Homology to Viral and Cellular Interferon Regulatory Factors 
Journal of Virology  2000;74(17):8194-8201.
The genome of the human herpesvirus 8 (HHV-8) contains a cluster of open reading frames (ORFs) encoding proteins with homology to the cellular transcription factors of the interferon regulatory factor (IRF) family. Two of these homologues, vIRF-1 and vIRF-2, were previously identified and functionally analyzed. In this study, we have characterized a novel gene, designated vIRF-3, encoded within the previously predicted ORF K10.5 and our newly identified ORF K10.6. Northern blotting of RNA extracted from BCBL-1 cells with a vIRF-3-specific probe and reverse transcription-PCR analyses revealed a single transcript of 2.2 kb with a splice present in the coding region. The vIRF-3 mRNA levels in BCBL-1 cells were increased upon 12-O-tetradecanoylphorbol-13-acetate treatment, with kinetics of expression similar to those of the early immediate genes. The vIRF-3 ORF encodes a 73-kDa protein with homology to cellular IRF-4 and HHV-8-encoded vIRF-2 and K11. In transient transfection assays with the IFNACAT reporter, vIRF-3 functioned as a dominant-negative mutant of both IRF-3 and IRF-7 and inhibited virus-mediated transcriptional activity of the IFNA promoter. Similarly, the overexpression of vIRF-3 in mouse L929 cells resulted in inhibition of virus-mediated synthesis of biologically active interferons. These results suggest that by targeting IRF-3 and IRF-7, which play a critical role in the activation of alpha/beta interferon (IFN) genes, HHV-8 has evolved a mechanism by which it directly subverts the functions of IRFs and down-regulates the induction of the IFN genes that are important components of the innate immunity.
PMCID: PMC112355  PMID: 10933732
11.  Synoviocyte innate responses: II. Pivotal role of interferon regulatory factor 3 
Introduction
Innate immune responses likely contribute to synovial inflammation in rheumatoid arthritis (RA). Of the innate receptors implicated in RA, TLR3 activates several signaling cascades, including -interferon regulatory factors 3 and 7 (IRF), resulting in production of viral-stress IFN-inducible genes. The present study was designed to investigate the contributions of IRF3 and IRF7 to the type I IFN response as well as the expression of other cytokines, chemokines, and degradative enzymes in synoviocytes.
Methods
Fibroblast-like synoviocytes (FLS) were stimulated with poly (I-C) after transfection with IRF3 or IRF7 siRNA to knockdown transcription factor expression. Western blots, luciferase assay after transfection with reporter constructs, Q-PCR, and AP-1 DNA binding ELISA was performed to evaluate the role of IRF3 and IRF7 in poly (I-C)-induced signaling and synoviocyte gene expression.
Results
IRF3 and IRF7 knockdown showed that IRF3 regulates IFN-stimulated response element (ISRE) promoter activity as well as IFNβ, IRF5, IRF7, RANTES, IP-10, MCP-1, and MIP1α gene expression in response to poly (I-C). IRF7 knockdown modestly decreased a subset of genes and ISRE activity, although the results were not significant. Surprisingly, IRF3 knockdown almost completely blocked expression of additional genes in which the ISRE is not traditionally considered a dominant promoter site in FLS, including MMP3, MMP9, IL-6 and IL-8. We then investigated a possible role for IRF3 in c-Jun activation and AP-1 binding because its promoter site is present in all four of the non-IFN regulated genes. IRF3 deficiency significantly decreased AP-1 binding of activated c-Jun compared with control.
Conclusions
In contrast to immune cells, IRF3 rather than IRF7 regulates TLR3-mediated type I IFN responses in human synoviocytes. IRF3 activates IFN-response gene expression by increasing ISRE promoter activity. In addition, IRF3 regulates other cytokines, chemokines, and MMPs through a novel mechanism that involves c-Jun and the AP-1 promoter site. Because the signaling pathway modulated by IRF3 plays a crucial role in synoviocytes, targeting IRF3 represents a potential approach to suppress diverse mediators while limiting suppression of IRF7-mediated immune responses.
doi:10.4049/jimmunol.0903944
PMCID: PMC2913682  PMID: 20483755
Rheumatoid arthritis; signal transduction; transcription factors; interferon
12.  The Structure of Classical Swine Fever Virus Npro: A Novel Cysteine Autoprotease and Zinc-Binding Protein Involved in Subversion of Type I Interferon Induction 
PLoS Pathogens  2013;9(10):e1003704.
Pestiviruses express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. The pestivirus N-terminal protease (Npro) is a cysteine autoprotease that cleaves between its own C-terminus and the N-terminus of the core protein. Due to its unique sequence and catalytic site, it forms its own cysteine protease family C53. After self-cleavage, Npro is no longer active as a protease. The released Npro suppresses the induction of the host's type-I interferon-α/β (IFN-α/β) response. Npro binds interferon regulatory factor-3 (IRF3), the key transcriptional activator of IFN-α/β genes, and promotes degradation of IRF3 by the proteasome, thus preventing induction of the IFN-α/β response to pestivirus infection. Here we report the crystal structures of pestivirus Npro. Npro is structurally distinct from other known cysteine proteases and has a novel “clam shell” fold consisting of a protease domain and a zinc-binding domain. The unique fold of Npro allows auto-catalysis at its C-terminus and subsequently conceals the cleavage site in the active site of the protease. Although many viruses interfere with type I IFN induction by targeting the IRF3 pathway, little information is available regarding structure or mechanism of action of viral proteins that interact with IRF3. The distribution of amino acids on the surface of Npro involved in targeting IRF3 for proteasomal degradation provides insight into the nature of Npro's interaction with IRF3. The structures thus establish the mechanism of auto-catalysis and subsequent auto-inhibition of trans-activity of Npro, and its role in subversion of host immune response.
Author Summary
Mammalian cells respond to viral infection by inducing an innate immune response involving interferon-α/β that mediates cellular antiviral defenses. Viruses, in turn, have evolved mechanisms to counter the host's innate immune response by inhibiting the interferon response. Pestiviruses use the virally encoded N-terminal protease (Npro) to suppress interferon-α/β induction. Npro first cleaves itself off from the viral polyprotein using its own cysteine protease activity. Released Npro then interacts with interferon regulatory factor-3 (IRF3), a transcriptional activator of interferon-β, and induces proteasome-mediated degradation of IRF3. We have determined the crystal structure of Npro from classical swine fever virus. Npro has a unique protease fold consisting of two domains. The N-terminal domain carries the protease active site and has the C-terminus, the auto-cleavage site, bound in the active site. Thus, following auto-cleavage at the C-terminus, Npro obstructs the catalytic site preventing further activity, making the protease active only once in its lifetime. The C-terminal domain carries a zinc-binding site that is required for interaction with IRF3. Surface mapping of the Npro residues essential for subversion of interferon induction provides insight into the interaction with IRF3 and its subsequent degradation. To our knowledge, this is the first structure of a direct IRF3 antagonist.
doi:10.1371/journal.ppat.1003704
PMCID: PMC3798407  PMID: 24146623
13.  The IRF-3 Transcription Factor Mediates Sendai Virus-Induced Apoptosis 
Journal of Virology  2000;74(8):3781-3792.
Virus infection of target cells can result in different biological outcomes: lytic infection, cellular transformation, or cell death by apoptosis. Cells respond to virus infection by the activation of specific transcription factors involved in cytokine gene regulation and cell growth control. The ubiquitously expressed interferon regulatory factor 3 (IRF-3) transcription factor is directly activated following virus infection through posttranslational modification. Phosphorylation of specific C-terminal serine residues results in IRF-3 dimerization, nuclear translocation, and activation of DNA-binding and transactivation potential. Once activated, IRF-3 transcriptionally up regulates alpha/beta interferon genes, the chemokine RANTES, and potentially other genes that inhibit viral infection. We previously generated constitutively active [IRF-3(5D)] and dominant negative (IRF-3 ΔN) forms of IRF-3 that control target gene expression. In an effort to characterize the growth regulatory properties of IRF-3, we observed that IRF-3 is a mediator of paramyxovirus-induced apoptosis. Expression of the constitutively active form of IRF-3 is toxic, preventing the establishment of stably transfected cells. By using a tetracycline-inducible system, we show that induction of IRF-3(5D) alone is sufficient to induce apoptosis in human embryonic kidney 293 and human Jurkat T cells as measured by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay, and analysis of DNA content by flow cytometry. Wild-type IRF-3 expression augments paramyxovirus-induced apoptosis, while expression of IRF-3 ΔN blocks virus-induced apoptosis. In addition, we demonstrate an important role of caspases 8, 9, and 3 in IRF-3-induced apoptosis. These results suggest that IRF-3, in addition to potently activating cytokine genes, regulates apoptotic signalling following virus infection.
PMCID: PMC111887  PMID: 10729153
14.  An incoherent regulatory network architecture that orchestrates B cell diversification in response to antigen signaling 
B cell receptor signaling controls the expression of IRF-4, a transcription factor required for B cell differentiation. This study shows that IRF-4 regulates divergent B cell fates via a ‘kinetic-control' mechanism that determines the duration of a transient developmental state.
The intensity of signaling through the B cell receptor controls the level of expression of IRF-4, a transcription factor required for B cell differentiation. The rate of IRF-4 production dictates the extent of antibody gene diversification that B cells undergo upon antigen encounter before differentiating into antibody-secreting plasma cells.Computational modeling and experimental analyses substantiate a model, whereby IRF-4 regulates B cell fate trajectories via a ‘kinetic-control' mechanism.Kinetic control is a process by which B cells pass through an obligate state of variable duration that sets the degree of cellular diversification prior to their terminal differentiation.An incoherent regulatory network architecture, within which IRF-4 is embedded, is the basis for realization of kinetic control.
The generation of a diverse set of pathogen-specific antibodies, with differing affinities and effector functions, by B lymphocytes is essential for efficient protection from many microorganisms. Antibody gene diversification in B cells is mediated by two molecular processes termed class-switch recombination and somatic hypermutation (CSR/SHM) (F1A). The former enables the generation of antibodies with the same antigen-binding specificity, but different effector domains, whereas the latter results in a repertoire of antibodies with a range of affinities for a given antigen containing the same effector domain. CSR/SHM occurs in antigen-activated B cells before their terminal differentiation into plasma cells. The transcription factor IRF-4 is required for CSR/SHM as well as plasma-cell differentiation, with its highest levels of expression being necessary for the latter. IRF-4 acts in the context of a network of regulators that include Blimp-1, Pax5, Bach2 and Bcl-6 (F1B). Despite extensive characterization of these individual factors, how the network responds to sensing of antigen by the B cell antigen receptor (BCR, antibody molecule expressed on cell surface) to regulate the extent of antibody gene diversification and plasma-cell differentiation remains to be addressed.
To address this issue, we assemble a computational model. The model reveals two contrasting scenarios that can underlie B cell fate dynamics. In one case, the initial rate of IRF-4 production controls a binary cell fate choice that involves either going to the CSR/SHM state or to the plasma-cell state; the time spent in the CSR state is relatively insensitive to the initial rate of IRF-4 production (herein called ‘basic bistability'). In the other case, IRF-4 drives all cells through a transient CSR/SHM state, but the initial rate of IRF-4 production sets its duration (‘kinetic control'). Both scenarios predict that increasing the initial rate of IRF-4 production favors the generation of plasma cells at the expense of CSR/SHM, but they differ fundamentally with respect to the underlying gene expression patterns.
To distinguish between these two scenarios experimentally, we utilize two different genetic models. The first involves the B1-8i transgenic mouse whose B cells express a rearranged V187.2 VDJ Ig heavy chain gene segment that is specific for the hapten nitrophenol (NP). The second is a newly developed mouse model that allows exogenous control of IRF-4 expression in naive primary B cells using a tet-inducible allele. Using these models, we show that (i) BCR signal strength sets the initial rate of IRF-4 accumulation and (ii) the concentration of IRF-4 is sensed by an incoherent gene regulatory network architecture to regulate the extent of CSR/SHM prior to plasma-cell differentiation. Our results are consistent with the ‘kinetic-control model' in which the levels of BCR-induced IRF-4 expression control the duration of an obligate CSR/SHM state that enables B cell diversification before terminal differentiation into plasma cells. Evidence for the transient CSR/SHM state is corroborated by both patterns of gene expression and the presence of AID-dependent mutations in individual non-switched plasmablasts.
Our results provide a molecular framework for understanding how B cells balance the competing demands for Ig CSR and SHM with the secretion of antibodies during humoral immune responses. The key feature of the network architecture that allows IRF-4 to coordinate the two competing states of gene expression in a temporal manner is that it simultaneously but asymmetrically activates both sides of a bistable mutual repression circuit. Because the two effects of the primary regulator antagonize each other, we describe the circuit as being based on an ‘incoherent' regulatory motif. Other incoherent regulatory motifs in varied biological systems are also associated with the acquisition of transient cell states, and we consider how the kinetic-control mechanism proposed by us could more generally serve to translate developmental cues into elaborate morphogenetic patterns.
The B-lymphocyte lineage is a leading system for analyzing gene regulatory networks (GRNs) that orchestrate distinct cell fate transitions. Upon antigen recognition, B cells can diversify their immunoglobulin (Ig) repertoire via somatic hypermutation (SHM) and/or class switch DNA recombination (CSR) before differentiating into antibody-secreting plasma cells. We construct a mathematical model for a GRN underlying this developmental dynamic. The intensity of signaling through the Ig receptor is shown to control the bimodal expression of a pivotal transcription factor, IRF-4, which dictates B cell fate outcomes. Computational modeling coupled with experimental analysis supports a model of ‘kinetic control', in which B cell developmental trajectories pass through an obligate transient state of variable duration that promotes diversification of the antibody repertoire by SHM/CSR in direct response to antigens. More generally, this network motif could be used to translate a morphogen gradient into developmental inductive events of varying time, thereby enabling the specification of distinct cell fates.
doi:10.1038/msb.2011.25
PMCID: PMC3130558  PMID: 21613984
BCR signal strength; bistability; gene regulatory network; ghost of a fixed point; Irf4
15.  Functional Analysis of a Dominant Negative Mutation of Interferon Regulatory Factor 5 
PLoS ONE  2009;4(5):e5500.
Background
Interferon regulatory factor (IRF) family members have been implicated as critical transcription factors that function in immune response, hematopoietic differentiation and cell growth regulation. Activation of IRF-5 results in the production of pro-inflammatory cytokines such as TNFα, IL6 and IL12p40, as well as type I interferons.
Methodology/Principal Findings
In this study, we identify a G202C (position relative to translation start codon) missense-mutation transcript of IRF-5 in transformed B and T cell lines, which were either infected or non-infected by viruses, and peripheral blood from ATL or CLL patients. The mutated transcript encodes a novel protein in which the sixty-eighth amino acid, Alanine, is substituted by Proline (IRF-5P68) in the DNA binding domain of IRF-5. IRF-5P68 phenotype results in a complete loss of its DNA-binding activity and functions as a dominant negative molecule through interacting with wild type IRF-5. Co-expression of IRF-5P68 inhibits MyD88-mediated IRF-5 transactivation. Moreover, Toll-like receptor (TLR)-dependent IL6 and IL12P40 production induced by lipopolysaccharide (LPS), R837 or CpG ODN 1826 was reduced in IRF-5 (P68) expressing cells as compared to the control cells.
Conclusion
IRF-5P68 acts as a dominant negative regulator that interferes with IRF-5-mediated production of pro-inflammatory cytokines. The functional characterization of the novel IRF-5 mutant in transformed B and T cell lines and in ATL and CLL patients may lead to a better understanding of the role of these transcriptional regulators in hematopoietic malignancies.
doi:10.1371/journal.pone.0005500
PMCID: PMC2677155  PMID: 19430534
16.  Structural and Functional Analysis of Interferon Regulatory Factor 3: Localization of the Transactivation and Autoinhibitory Domains 
Molecular and Cellular Biology  1999;19(4):2465-2474.
The interferon regulatory factor 3 (IRF-3) gene encodes a 55-kDa protein which is expressed constitutively in all tissues. In unstimulated cells, IRF-3 is present in an inactive cytoplasmic form; following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues located in the carboxy terminus. Virus-induced phosphorylation of IRF-3 leads to cytoplasmic to nuclear translocation of phosphorylated IRF-3, association with the transcriptional coactivator CBP/p300, and stimulation of DNA binding and transcriptional activities of virus-inducible genes. Using yeast and mammalian one-hybrid analysis, we now demonstrate that an extended, atypical transactivation domain is located in the C terminus of IRF-3 between amino acids (aa) 134 and 394. We also show that the C-terminal domain of IRF-3 located between aa 380 and 427 participates in the autoinhibition of IRF-3 activity via an intramolecular association with the N-terminal region between aa 98 and 240. After Sendai virus infection, an intermolecular association between IRF-3 proteins is detected, demonstrating a virus-dependent formation of IRF-3 homodimers; this interaction is also observed in the absence of virus infection with a constitutively activated form of IRF-3. Substitution of the C-terminal Ser-Thr phosphorylation sites with the phosphomimetic Asp in the region ISNSHPLSLTSDQ between amino acids 395 and 407 [IRF-3(5D)], but not the adjacent S385 and S386 residues, generates a constitutively activated DNA binding form of IRF-3. In contrast, substitution of S385 and S386 with either Ala or Asp inhibits both DNA binding and transactivation activities of the IRF-3(5D) protein. These studies thus define the transactivation domain of IRF-3, two domains that participate in the autoinhibition of IRF-3 activity, and the regulatory phosphorylation sites controlling IRF-3 dimer formation, DNA binding activity, and association with the CBP/p300 coactivator.
PMCID: PMC84039  PMID: 10082512
17.  GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. 
Molecular and Cellular Biology  1997;17(5):2735-2744.
After binding to enhancer elements, transcription factors require transcriptional coactivator proteins to mediate their stimulation of transcription initiation. A search for possible coactivators for steroid hormone receptors resulted in identification of glucocorticoid receptor interacting protein 1 (GRIP1). The complete coding sequence for GRIP1, isolated from a mouse brain cDNA library, contains an open reading frame of 1,462 codons. GRIP1 is the probable ortholog of the subsequently identified human protein transcription intermediary factor 2 (TIF2) and is also partially homologous to steroid receptor coactivator 1 (SRC-1). The full-length GRIP1 interacted with the hormone binding domains (HBDs) of all five steroid receptors in a hormone-dependent manner and also with HBDs of class II nuclear receptors, including thyroid receptor alpha, vitamin D receptor, retinoic acid receptor alpha, and retinoid X receptor alpha. In contrast to agonists, glucocorticoid antagonists did not promote interaction between the glucocorticoid receptor and GRIP1. In yeast cells, GRIP1 dramatically enhanced the transcriptional activation function of proteins containing the HBDs of any of the above-named receptors fused to the GAL4 DNA binding domain and thus served as a transcriptional coactivator for them. This finding contrasts with previous reports of TIF2 and SRC-1, which in mammalian cells enhanced the transactivation activities of only a subset of the steroid and nuclear receptors that they physically interacted with. GRIP1 also enhanced the hormone-dependent transactivation activity of intact glucocorticoid receptor, estrogen receptor, and mineralocorticoid receptor. Experiments with glucocorticoid receptor truncation and point mutants indicated that GRIP1 interacted with and enhanced the activity of the C-terminal AF-2 but not the N-terminal AF-1 transactivation domain of the glucocorticoid receptor. These results demonstrate directly that AF-1 and AF-2 domains accomplish their transactivation activities through different mechanisms: AF-2 requires GRIP1 as a coactivator, but AF-1 does not.
PMCID: PMC232124  PMID: 9111344
18.  Interferon Regulatory Factor 8 Regulates Pathways for Antigen Presentation in Myeloid Cells and during Tuberculosis 
PLoS Genetics  2011;7(6):e1002097.
IRF8 (Interferon Regulatory Factor 8) plays an important role in defenses against intracellular pathogens, including several aspects of myeloid cells function. It is required for ontogeny and maturation of macrophages and dendritic cells, for activation of anti-microbial defenses, and for production of the Th1-polarizing cytokine interleukin-12 (IL-12) in response to interferon gamma (IFNγ) and protection against infection with Mycobacterium tuberculosis. The transcriptional programs and cellular pathways that are regulated by IRF8 in response to IFNγ and that are important for defenses against M. tuberculosis are poorly understood. These were investigated by transcript profiling and chromatin immunoprecipitation on microarrays (ChIP-chip). Studies in primary macrophages identified 368 genes that are regulated by IRF8 in response to IFNγ/CpG and that behave as stably segregating expression signatures (eQTLs) in F2 mice fixed for a wild-type or mutant allele at IRF8. A total of 319 IRF8 binding sites were identified on promoters genome-wide (ChIP-chip) in macrophages treated with IFNγ/CpG, defining a functional G/AGAAnTGAAA motif. An analysis of the genes bearing a functional IRF8 binding site, and showing regulation by IFNγ/CpG in macrophages and/or in M. tuberculosis-infected lungs, revealed a striking enrichment for the pathways of antigen processing and presentation, including multiple structural and enzymatic components of the Class I and Class II MHC (major histocompatibility complex) antigen presentation machinery. Also significantly enriched as IRF8 targets are the group of endomembrane- and phagosome-associated small GTPases of the IRG (immunity-related GTPases) and GBP (guanylate binding proteins) families. These results identify IRF8 as a key regulator of early response pathways in myeloid cells, including phagosome maturation, antigen processing, and antigen presentation by myeloid cells.
Author Summary
IRF8 is a member of the Interferon Regulatory Factor family that is expressed in myeloid cells such as macrophages and dendritic cells and that activates or represses gene transcription upon stimulation with interferon gamma (IFNγ), lipopolysaccharide (LPS), and other microbial stimuli. IRF8 plays an important role in several aspects of myeloid cells, including differentiation and maturation of early progenitor cells, expression of intrinsic anti-microbial defenses, and production of the interleukin-12 (IL12) cytokine, which is essential for priming of early T cell– mediated immune response. IRF8 mutant mice are susceptible to a number of intracellular infections including pulmonary tuberculosis. The transcriptional and cellular pathways regulated by IRF8 and essential for resistance to infections were studied by a combination of genome-wide methods, including transcriptional profiling and chromatin immunoprecipitation (ChIP-chip). These studies identified phagosome maturation, antigen processing, and antigen presentation as critical pathways in early host–pathogen interactions regulated by IRF8 in macrophages exposed to IFNγ/CpG and in lung tissues infected with Mycobacterium tuberculosis.
doi:10.1371/journal.pgen.1002097
PMCID: PMC3121741  PMID: 21731497
19.  The Type I Interferon Signaling Pathway Is a Target for Glucocorticoid Inhibition ▿  
Molecular and Cellular Biology  2010;30(19):4564-4574.
Type I interferon (IFN) is essential for host defenses against viruses; however, dysregulated IFN signaling is causally linked to autoimmunity, particularly systemic lupus erythematosus. Autoimmune disease treatments rely on glucocorticoids (GCs), which act via the GC receptor (GR) to repress proinflammatory cytokine gene transcription. Conversely, cytokine signaling through cognate Jak/STAT pathways is reportedly unaffected or even stimulated by GR. Unexpectedly, we found that GR dramatically inhibited IFN-stimulated gene (ISG) expression in macrophages. The target of inhibition, the heterotrimeric STAT1-STAT2-IRF9 (ISGF3) transcription complex, utilized the GR cofactor GRIP1/TIF2 as a coactivator. Consequently, GRIP1 knockdown, genetic ablation, or depletion by GC-activated GR attenuated ISGF3 promoter occupancy, preinitiation complex assembly, and ISG expression. Furthermore, this regulatory loop was restricted to cell types such as macrophages expressing the GRIP1 protein at extremely low levels, and pharmacological disruption of the GR-GRIP1 interaction or transient introduction of GRIP1 restored RNA polymerase recruitment to target ISGs and the subsequent IFN response. Thus, type I IFN is a cytokine uniquely controlled by GR at the levels of not only production but also signaling through antagonism with the ISGF3 effector function, revealing a novel facet of the immunosuppressive properties of GCs.
doi:10.1128/MCB.00146-10
PMCID: PMC2950533  PMID: 20679482
20.  Human Herpesvirus 8 Interferon Regulatory Factor-Mediated BH3-Only Protein Inhibition via Bid BH3-B Mimicry 
PLoS Pathogens  2012;8(6):e1002748.
Viral replication efficiency is in large part governed by the ability of viruses to counteract pro-apoptotic signals induced by infection of host cells. For HHV-8, viral interferon regulatory factor-1 (vIRF-1) contributes to this process in part via inhibitory interactions with BH3-only protein (BOP) Bim, recently identified as an interaction partner of vIRF-1. Here we recognize that the Bim-binding domain (BBD) of vIRF-1 resembles a region (BH3-B) of Bid, another BOP, which interacts intramolecularly with the functional BH3 domain of Bid to inhibit it pro-apoptotic activity. Indeed, vIRF-1 was found to target Bid in addition to Bim and to interact, via its BBD region, with the BH3 domain of each. In functional assays, BBD could substitute for BH3-B in the context of Bid, to suppress Bid-induced apoptosis in a BH3-binding-dependent manner, and vIRF-1 was able to protect transfected cells from apoptosis induced by Bid. While vIRF-1 can mediate nuclear sequestration of Bim, this was not the case for Bid, and inhibition of Bid and Bim by vIRF-1 could occur independently of nuclear localization of the viral protein. Consistent with this finding, direct BBD-dependent inactivation by vIRF-1 of Bid-induced mitochondrial permeabilization was demonstrable in vitro and isolated BBD sequences were also active in this assay. In addition to Bim and Bid BH3 domains, BH3s of BOPs Bik, Bmf, Hrk, and Noxa also were found to bind BBD, while those of both pro- and anti-apoptotic multi-BH domain Bcl-2 proteins were not. Finally, the significance of Bid to virus replication was demonstrated via Bid-depletion in HHV-8 infected cells, which enhanced virus production. Together, our data demonstrate and characterize BH3 targeting and associated inhibition of BOP pro-apoptotic activity by vIRF-1 via Bid BH3-B mimicry, identifying a novel mechanism of viral evasion from host cell defenses.
Author Summary
Viruses possess mechanisms of subverting host cell defenses against infection and virus replication; these mechanisms are essential to the virus life cycle. Here, we identify and characterize a novel mechanism of HHV-8 mediated inhibition of virus-induced programmed cell death (apoptosis). This function is specified by viral interferon regulator factor homologue vIRF-1, which binds to and directly inhibits pro-death activities of so-called BH3-only proteins (BOPs), induced and activated by stress signals such as those occurring in infected cells. The BH3 domains of BOPs mediate their pro-apoptotic functions, and it is these domains that are targeted by vIRF-1, via a region resembling a BH3-interacting and -inhibitory domain, termed BH3-B, present in one of the vIRF-1 targeted BOPs, Bid. The targeted BOP BH3 domains share characteristic and conserved features. As shown previously for Bim, depletion of Bid leads to enhanced HHV-8 productive replication, demonstrating that Bid, also, is a biologically significant negative regulator of virus replication and suggesting that its control by vIRF-1 is of functional importance. To our knowledge, this is the first report of viral targeting and inhibition of BOP activity via Bid BH3-B mimicry; our studies therefore expand the known mechanisms of viral evasion from antiviral defenses of the host.
doi:10.1371/journal.ppat.1002748
PMCID: PMC3369933  PMID: 22685405
21.  Inhibition of Interferon Regulatory Factor 7 (IRF7)-Mediated Interferon Signal Transduction by the Kaposi's Sarcoma-Associated Herpesvirus Viral IRF Homolog vIRF3▿  
Journal of Virology  2007;81(15):8282-8292.
Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway that is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate the host IFN-mediated immune response. Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologs of the cellular IRFs, called vIRFs. Here, we report a novel immune evasion mechanism of KSHV vIRF3 to block cellular IRF7-mediated innate immunity in response to viral infection. KSHV vIRF3 specifically interacts with either the DNA binding domain or the central IRF association domain of IRF7, and this interaction leads to the inhibition of IRF7 DNA binding activity and, therefore, suppression of alpha interferon (IFN-α) production and IFN-mediated immunity. Remarkably, the central 40 amino acids of vIRF3, containing the double α helix motifs, are sufficient not only for binding to IRF7, but also for inhibiting IRF7 DNA binding activity. Consequently, the expression of the double α helix motif-containing peptide effectively suppresses IRF7-mediated IFN-α production. This demonstrates a remarkably efficient means of viral avoidance of host antiviral activity.
doi:10.1128/JVI.00235-07
PMCID: PMC1951281  PMID: 17522209
22.  The Viral Interferon Regulatory Factors of Kaposi's Sarcoma-Associated Herpesvirus Differ in Their Inhibition of Interferon Activation Mediated by Toll-Like Receptor 3 
Journal of Virology  2013;87(2):798-806.
Kaposi's sarcoma-associated herpesvirus (KSHV) infection is correlated with three human malignancies and can establish lifelong latent infection in multiple cell types within its human host. In order to establish and maintain infection, KSHV utilizes multiple mechanisms to evade the host immune response. One such mechanism is the expression of a family of genes with homology to cellular interferon (IFN) regulatory factors (IRFs), known as viral IRFs (vIRFs). We demonstrate here that KSHV vIRF1, -2, and -3 have a differential ability to block type I interferon signaling mediated by Toll-like receptor 3 (TLR3), a receptor we have previously shown to be activated upon KSHV infection. vIRF1, -2, and -3 inhibited TLR3-driven activation of IFN transcription reporters. However, only vIRF1 and vIRF2 inhibited increases in both IFN-β message and protein levels following TLR3 activation. The expression of vIRF1 and vIRF2 also allowed for increased replication of a virus known to activate TLR3 signaling. Furthermore, vIRF1 and vIRF2 may block TLR3-mediated signaling via different mechanisms. Altogether, this report indicates that vIRFs are able to block IFN mediated by TLRs but that each vIRF has a unique function and mechanism for blocking antiviral IFN responses.
doi:10.1128/JVI.01851-12
PMCID: PMC3554052  PMID: 23115281
23.  Virus-Dependent Phosphorylation of the IRF-3 Transcription Factor Regulates Nuclear Translocation, Transactivation Potential, and Proteasome-Mediated Degradation 
Molecular and Cellular Biology  1998;18(5):2986-2996.
The interferon regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the alpha beta interferon (IFN-α/β) gene promoters, as well as the interferon-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter. IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. In the present study, we demonstrate that following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, which are located in the carboxy terminus of IRF-3. A combination of IRF-3 deletion and point mutations localized the inducible phosphorylation sites to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407; point mutation of residues Ser-396 and Ser-398 eliminated virus-induced phosphorylation of IRF-3 protein, although residues Ser-402, Thr-404, and Ser-405 were also targets. Phosphorylation results in the cytoplasm-to-nucleus translocation of IRF-3, DNA binding, and increased transcriptional activation. Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements. Phosphorylation also appears to represent a signal for virus-mediated degradation, since the virus-induced turnover of IRF-3 was prevented by mutation of the IRF-3 Ser-Thr cluster or by proteasome inhibitors. Interestingly, virus infection resulted in the association of IRF-3 with the CREB binding protein (CBP) coactivator, as detected by coimmunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins. Mutation of residues Ser-396 and Ser-398 in IRF-3 abrogated its binding to CBP. These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes.
PMCID: PMC110678  PMID: 9566918
24.  Classical Swine Fever Virus Npro Interacts with Interferon Regulatory Factor 3 and Induces Its Proteasomal Degradation▿  
Journal of Virology  2007;81(7):3087-3096.
Viruses have evolved a multitude of strategies to subvert the innate immune system by interfering with components of the alpha/beta interferon (IFN-α/β) induction and signaling pathway. It is well established that the pestiviruses prevent IFN-α/β induction in their primary target cells, such as epitheloidal and endothelial cells, macrophages, and conventional dendritic cells, a phenotype mediated by the viral protein Npro. Central players in the IFN-α/β induction cascade are interferon regulatory factor 3 (IRF3) and IRF7. Recently, it was proposed that classical swine fever virus (CSFV), the porcine pestivirus, induced the loss of IRF3 by inhibiting the transcription of IRF3 mRNA. In the present study, we show that endogenous IRF3 and IRF3 expressed from a cytomegalovirus (CMV) promoter are depleted in the presence of CSFV by means of Npro, while CSFV does not inhibit CMV promoter-driven protein expression. We also demonstrate that CSFV does not reduce the transcriptional activity of the IRF3 promoter and does not affect the stability of IRF3 mRNA. In fact, CSFV Npro induces proteasomal degradation of IRF3, as demonstrated by proteasome inhibition studies. Furthermore, Npro coprecipitates with IRF3, suggesting that the proteasomal degradation of IRF3 is induced by a direct or indirect interaction with Npro. Finally, we show that Npro does not downregulate IRF7 expression.
doi:10.1128/JVI.02032-06
PMCID: PMC1866024  PMID: 17215286
25.  Cytokines Alter Glucocorticoid Receptor Phosphorylation in Airway Cells 
Corticosteroid insensitivity (CSI) represents a profound challenge in managing patients with asthma. We recently demonstrated that short exposure of airway smooth muscle cells (ASMCs) to proasthmatic cytokines drastically reduced their responsiveness to glucocorticoids (GCs), an effect that was partially mediated via interferon regulatory factor-1, suggesting the involvement of additional mechanisms (Am J Respir Cell Mol Biol 2008;38:463–472). Although GC receptor (GR) can be phosphorylated at multiple serines in the N-terminal region, the major phosphorylation sites critical for GR transcriptional activity are serines 211 (Ser211) and 226 (Ser226). We tested the novel hypothesis that cytokine-induced CSI in ASMCs is due to an impaired GR phosphorylation. Cells were treated with TNF-α (10 ng/ml) and IFN-γ (500 UI/ml) for 6 hours and/or fluticasone (100 nm) added 2 hours before. GR was constitutively phosphorylated at Ser226 but not at Ser211 residues. Cytokines dramatically suppressed fluticasone-induced phosphorylation of GR on Ser211 but not on Ser226 residues while increasing the expression of Ser/Thr protein phosphatase (PP)5 but not that of PP1 or PP2A. Transfection studies using a reporter construct containing GC responsive elements showed that the specific small interfering RNA–induced mRNA knockdown of PP5, but not that of PP1 or PP2A, partially prevented the cytokine suppressive effects on GR-meditated transactivation activity. Similarly, cytokines failed to inhibit GC-induced GR-Ser211 phosphorylation when expression of PP5 was suppressed. We propose that the novel mechanism that proasthmatic cytokine-induced CSI in ASMCs is due, in part, to PP5-mediated impairment of GR-Ser211 phosphorylation.
doi:10.1165/rcmb.2011-0364OC
PMCID: PMC3488623  PMID: 22592921
serine/threonine protein phosphatase; airway smooth muscle; asthma; corticosteroid insensitivity; airway remodeling

Results 1-25 (1092933)