Molecular characterization of interferon regulatory factor 1 in Bubalus bubalis

Interferon regulatory factor 1 (IRF1) is functionally diverse in the regulation of immune response and is considered to be an important candidate gene for studying disease susceptibility in mammals. In this paper, we characterized the whole sequence of the IRF1 gene in river buffalo (Bubalus bubalis) and compared genomic and the amino acid sequences between different species. The buffalo IRF1 gene was 7099 bp long and organized into 10 exons and nine introns. Its molecular structure showed exactly the same number of exons (10) and introns (nine) in bovids, mice, horses, humans, and chickens. However, rats did not have exon 5, but had the largest exon 4, which suggests that exon 5 was incorporated into exon 4. The coding and the amino acid sequences of the gene showed that identity varied from 73 to 99% at the coding sequence level and from 61 to 100% at the amino acid level when compared with other mammals and chickens. Comparative analysis of the gene sequence between two different buffalo breeds, Murrah and Mediterranean, revealed six potential SNPs that are primarily located in the 5' and 3'UTRs.

Inhibition of human immunodeficiency virus type 1 and vaccinia virus infection by a dominant negative factor of the interferon regulatory factor family expressed in monocytic cells.

ICSBP is a member of the interferon (IFN) regulatory factor (IRF) family that regulates expression of type I interferon (IFN) and IFN-regulated genes. To study the role of the IRF family in viral infection, a cDNA for the DNA-binding domain (DBD) of ICSBP was stably transfected into U937 human monocytic cells. Clones that expressed DBD exhibited a dominant negative phenotype and did not elicit antiviral activity against vesicular stomatitis virus (VSV) infection upon IFN treatment. Most notably, cells expressing DBD were refractory to infection by vaccinia virus (VV) and human immunodeficiency virus type 1 (HIV-1). The inhibition of VV infection was attributed to defective virion assembly, and that of HIV-1 to low CD4 expression and inhibition of viral transcription in DBD clones. HIV-1 and VV were found to have sequences in their regulatory regions similar to the IFN-stimulated response element (ISRE) to which IRF family proteins bind. Accordingly, these viral sequences and a cellular ISRE bound a shared factor(s) expressed in U937 cells. These observations suggest a novel host-virus relationship in which the productive infection of some viruses is regulated by the IRF-dependent transcription pathway through the ISRE.

Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family.

Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs.

Arenavirus Nucleoprotein Targets Interferon Regulatory Factor-Activating Kinase IKKε.

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.

An interferon regulatory factor element controls the major immune modulator of murine cytomegalovirus

Immune modulation by herpesviruses, such as cytomegalovirus, is critical for the establishment of acute and persistent infection confronting a vigorous antiviral immune response of the host. Therefore, the action of immune-modulatory proteins has long been the subject of research, with the final goal to identify new strategies for antiviral therapy.rnIn the case of murine cytomegalovirus (mCMV), the viral m152 protein has been identified to play a major role in targeting components of both the innate and the adaptive immune system in terms of infected host-cell recognition in the effector phase of the antiviral immune response. On the one hand, it inhibits cell surface expression of RAE-1 and thereby prevents ligation of the activating natural killer (NK)-cell receptor NKG2D. On the other hand, it decreases cell surface expression of peptide-loaded MHC class I molecules thereby preventing antigen presentation to CD8 T cells. Ultimately, the outcome of CMV infection is determined by the interplay between viral and cellular factors.rnIn this context, the work presented here has revealed a novel and intriguing connection between viral m152 and cellular interferon (IFN), a key cytokine of the immune system: rnthe m152 promoter region contains an interferon regulatory factor element (IRFE) perfectly matching the consensus sequence of cellular IRFEs.rnThe biological relevance of this regulatory element was first suggested by sequence comparisons revealing its evolutionary conservation among various established laboratory strains of mCMV and more recent low-passage wild-derived virus isolates. Moreover, search of the mCMV genome revealed only three IRFE sites in the complete sequence. Importantly, the functionality of the IRFE in the m152 promoter was confirmed with the use of a mutant virus, representing a functional deletion of the IRFE, and its corresponding revertant virus. In particular, m152 gene expression was found to be inhibited in an IRFE-dependent manner in infected cells. Essentially, this inhibition proved to have a severe impact on the immune-modulatory function of m152, first demonstrated by a restored direct antigen presentation on infected cells for CD8 T-cell activation. Even more importantly, this effect of IRFE-mediated IFN signaling was validated in vivo by showing that the protective antiviral capacity of adoptively-transferred, antigen-specific CD8 T cells is also significantly restored by the IRFE-dependent inhibition of m152. Somewhat curious and surprising, the decrease in m152 protein simultaneously prevented an enhanced activation of NK cells in acute-infected mice, apparently independent of the RAE-1/NKG2D ligand/receptor interaction but rather due to reduced ‘missing-self’ recognition.rnTaken together, this work presents a so far unknown mechanism of IFN signaling to control mCMV immune modulation in acute infection.rnrn

Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes.

A family of interferon (IFN) regulatory factors (IRFs) have been shown to play a role in transcription of IFN genes as well as IFN-stimulated genes. We report the identification of a member of the IRF family which we have named IRF-3. The IRF-3 gene is present in a single copy in human genomic DNA. It is expressed constitutively in a variety of tissues and no increase in the relative steady-state levels of IRF-3 mRNA was observed in virus-infected or IFN-treated cells. The IRF-3 gene encodes a 50-kDa protein that binds specifically to the IFN-stimulated response element (ISRE) but not to the IRF-1 binding site PRD-I. Overexpression of IRF-3 stimulates expression of the IFN-stimulated gene 15 (ISG15) promoter, an ISRE-containing promoter. The murine IFNA4 promoter, which can be induced by IRF-1 or viral infection, is not induced by IRF-3. Expression of IRF-3 as a Gal4 fusion protein does not activate expression of a chloramphenicol acetyltransferase reporter gene containing repeats of the Gal4 binding sites, indicating that this protein does not contain the transcription transactivation domain. The high amino acid homology between IRF-3 and ISG factor 3 gamma polypeptide (ISGF3 gamma) and their similar binding properties indicate that, like ISGF3 gamma, IRF-3 may activate transcription by complex formation with other transcriptional factors, possibly members of the Stat family. Identification of this ISRE-binding protein may help us to understand the specificity in the various Stat pathways.

Prevalence and nonrandom distribution of exonic mutations in interferon regulatory factor 6 in 307 families with Van der Woude syndrome and 37 families with popliteal pterygium syndrome

Purpose: Interferon regulatory factor 6 encodes a member of the IRF family of transcription factors. Mutations in interferon regulatory factor 6 cause Van der Woude and popliteal pterygium syndrome, two related orofacial clefting disorders. Here, we compared and contrasted the frequency and distribution of exonic Mutations in interferon regulatory factor 6 between two large geographically distinct collections of families with Van der Woude and between one collection of families with popliteal pterygium syndrome. Methods: We performed direct sequence analysis of interferon regulatory factor 6 exons oil samples from three collections, two with Van der Woude and one with popliteal pterygium syndrome. Results: We identified mutations in interferon regulatory factor 6 exons in 68% of families in both Van der Woude collections and in 97% of families with popliteal pterygium syndrome. In sum, 106 novel disease-causing variants were found. The distribution of mutations in the interferon regulatory factor 6 exons in each collection was not random; exons 3, 4, 7, and 9 accounted for 80%. In the Van der Woude collections, the mutations were evenly divided between protein truncation and missense, whereas most mutations identified in the popliteal pterygium syndrome collection were missense. Further, the missense mutations associated with popliteal pterygium syndrome were localized significantly to exon 4, at residues that are predicted to bind directly to DNA. Conclusion: The nonrandom distribution of mutations in the interferon regulatory factor 6 exons suggests a two-tier approach for efficient mutation screens for interferon regulatory factor 6. The type and distribution of mutations are consistent with the hypothesis that Van der Woude is caused by haploinsufficiency of interferon regulatory factor 6. Oil the other hand, the distribution of popliteal pterygium syndrome-associated mutations suggests a different, though not mutually exclusive, effect oil interferon regulatory factor 6 function. Genet Med 2009:11(4):241-247.

Prevalence and nonrandom distribution of exonic mutations in interferon regulatory factor 6 in 307 families with Van der Woude syndrome and 37 families with popliteal pterygium syndrome

Purpose: Interferon regulatory factor 6 encodes a member of the IRF family of transcription factors. Mutations in interferon regulatory factor 6 cause Van der Woude and popliteal pterygium syndrome, two related orofacial clefting disorders. Here, we compared and contrasted the frequency and distribution of exonic Mutations in interferon regulatory factor 6 between two large geographically distinct collections of families with Van der Woude and between one collection of families with popliteal pterygium syndrome. Methods: We performed direct sequence analysis of interferon regulatory factor 6 exons oil samples from three collections, two with Van der Woude and one with popliteal pterygium syndrome. Results: We identified mutations in interferon regulatory factor 6 exons in 68% of families in both Van der Woude collections and in 97% of families with popliteal pterygium syndrome. In sum, 106 novel disease-causing variants were found. The distribution of mutations in the interferon regulatory factor 6 exons in each collection was not random; exons 3, 4, 7, and 9 accounted for 80%. In the Van der Woude collections, the mutations were evenly divided between protein truncation and missense, whereas most mutations identified in the popliteal pterygium syndrome collection were missense. Further, the missense mutations associated with popliteal pterygium syndrome were localized significantly to exon 4, at residues that are predicted to bind directly to DNA. Conclusion: The nonrandom distribution of mutations in the interferon regulatory factor 6 exons suggests a two-tier approach for efficient mutation screens for interferon regulatory factor 6. The type and distribution of mutations are consistent with the hypothesis that Van der Woude is caused by haploinsufficiency of interferon regulatory factor 6. Oil the other hand, the distribution of popliteal pterygium syndrome-associated mutations suggests a different, though not mutually exclusive, effect oil interferon regulatory factor 6 function. Genet Med 2009:11(4):241-247.

The human papillomavirus type 16 E7 protein binds human interferon regulatory factor-9 via a novel PEST domain required for transformation

It is critical that viruses are able to avoid the antiviral activities of interferon (IFN). We have shown previously that the human papillomavirus (HPV) is able to avoid IFN-alpha via interaction of the HPV-16 E7 protein with IFN regulatory factor-9 (IRF-9). Here, we investigated the details of the interaction using HPV-16 E7 peptide mapping to show that IRF-9 binds HPV-16 E7 in a domain encompassing amino acids 25-36. A closer examination of this region indicates this is a novel proline, glutamate, serine, and threonine-rich (PEST) domain, with a PEST score of 8.74. We have also mapped the region of interaction within IRF-9 and found that amino acids 354-393 play an important role in binding to HPV-16 E7. This region of IRF-9 encompasses the IRF association domain (IAD), a region important for protein-protein interaction central to IRF function. Finally, we used alanine-scanning mutagenesis to determine if E7-IRF-9 interaction was important for E7-mediated cellular transformation and found that the HPV-16 E7 mutants Y25A, E26A, S31A, S32A, and E35A, but not L28A and N29A, caused loss of transformation ability. Preliminary data suggest loss of IRF-9 interaction with E7 mutants correlated with transformation. Our work suggests E7- IRF- 9 interaction is important for the transforming ability of HPV-16 E7 and that HPV-16 E7 may interact with other IRF proteins that have IAD domains.

Toxoplasma gondii: The severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability

In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-gamma and iNOS expression in the CNS compared to BALB/c mice. The CNS of C578L/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite. (C) 2010 Elsevier Inc. All rights reserved.

Expression of interferon-gamma, interferon-alpha and related genes in individuals with Down syndrome and periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-β enhanceosome in vitro

The transcriptional activity of an in vitro assembled human interferon-β gene enhanceosome is highly synergistic. This synergy requires five distinct transcriptional activator proteins (ATF2/c-JUN, interferon regulatory factor 1, and p50/p65 of NF-κB), the high mobility group protein HMG I(Y), and the correct alignment of protein-binding sites on the face of the DNA double helix. Here, we investigate the mechanisms of enhanceosome-dependent transcriptional synergy during preinitiation complex assembly in vitro. We show that the stereospecific assembly of the enhanceosome is critical for the efficient recruitment of TFIIB into a template-committed TFIID-TFIIA-USA (upstream stimulatory activity complex) and for the subsequent recruitment of the RNA polymerase II holoenzyme complex. In addition, we provide evidence that recruitment of the holoenzyme by the enhanceosome is due, at least in part, to interactions between the enhanceosome and the transcriptional coactivator CREB, cAMP responsive element binding protein (CBP). These studies reveal a unique role of enhanceosomes in the cooperative assembly of the transcription machinery on the human interferon-β promoter.

Interleukin 2 activates STAT5 transcription factor (mammary gland factor) and specific gene expression in T lymphocytes.

Although prolactin and interleukin 2 (IL-2) can elicit distinct physiological responses, we have found that their signal pathways share a common signal transducer and activator of transcription, STAT5. STAT5 was originally identified as a mammary gland factor induced by prolactin in lactating breast cells. Here we demonstrate that STAT5 is activated after IL-2 stimulation of two responsive lymphocyte cell lines, Nb2 and YT. Activation of STAT5 is measured both by IL-2-induced tyrosine phosphorylation and by IL-2-induced DNA binding. The STAT5 DNA recognition site is the same as the interferon gamma-activated site (GAS) in the interferon regulatory factor 1 gene. We demonstrate that the GAS element is necessary and sufficient for transcriptional induction by both IL-2 and prolactin in T lymphocytes. These results indicate that the role of STAT5 in the regulation of gene expression is not restricted to mammary cells or to prolactin, but is an integral part of the signal pathway of a critical immunomodulatory cytokine, IL-2.

Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

BACKGROUND The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-gamma-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-gamma treatment in human melanoma cell lines. METHODS Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan(R) Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-gamma-treated cells. RESULTS Altered IFN-gamma mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-alpha led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-gamma treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-alpha treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-gamma in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-gamma signaling pathway. CONCLUSION We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-gamma signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-gamma-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation.

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortus Delta wbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. Delta wbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence. (C) 2010 Elsevier Ltd. All rights reserved.