Evidence for IRF-1-dependent gene expression deficiency in interferon unresponsive HepG2 cells.
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Induction of the antiproliferative and antiviral state by IFNs (type I and II) is dramatically impaired in HepG2 cells. We show here that RNase L, IDO, GBP-2 and iNOS genes normally expressed as a secondary response to IFN are no longer inducible in HepG2 cells, while induction of primary response genes (IRF-1, PKR, p48-ISGF3gamma, 2-5AS, 6-16 and p56-(trp)tRNA) are unaffected. On the basis of previous data implicating transcription factor IRF-1 in the induction of some IFN-induced genes, we tested the effects of transfecting an IRF-1 oligonucleotide antisense in HeLa cells and found specifically impaired IFN induction of secondary response genes (RNase L, IDO and GBP-2). This raised the possibility that IRF-1 was defective in HepG2 cells. However, some molecular and biochemical analyses reveal that IRF-1 is induced normally by IFNs and retains its normal size, cellular location, phosphorylation status and ability to bind the IDO promoter in vitro. Therefore, we conclude that although the primary response pathway is fully functional, some aspects of the secondary pathway involving IRF-1 (but not IRF-1 itself) are defective in HepG2 cells. It may be possible that the promoter region of these deficient HepG2-genes requires an unidentified transcription factor in addition to de novo IRF-1, which could be elicited by a cooperative activator. (+info)
Differential expression and distinct functions of IFN regulatory factor 4 and IFN consensus sequence binding protein in macrophages.
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IFN regulatory factor 4 (IRF4) and IFN consensus sequence binding protein (ICSBP) are highly homologous members of the growing family of IRF proteins. ICSBP expression is restricted to lymphoid and myeloid cells, whereas IRF4 expression has been reported to be lymphoid-restricted. We present evidence that primary murine and human macrophages express IRF4, thereby extending its range of expression to myeloid cells. Here, we provide a comparative analysis of IRF4 and ICSBP expression and function in distinct cell types. These IRF proteins can form specific complexes with the Ets-like protein PU.1, and can activate transcription via binding to PU.1/IRF composite sequences. EMSA analysis revealed that murine macrophages contained both IRF4/PU.1 and ICSBP/PU.1 complexes, analogous to B cells. Over-expression of ICSBP in these macrophages activated transcription of a PU.1/IRF-dependent promoter, whereas over-expression of IRF4 had no effect on this promoter. In contrast, over-expression of either IRF4 or ICSBP in both macrophages and NIH-3T3 fibroblasts suppressed transcription of the PU.1-independent H-2Ld MHC class I promoter. In NIH-3T3 fibroblasts, IRF4 and ICSBP also synergized with exogenous PU.1 to activate transcription of a PU.1/IRF-dependent promoter. Furthermore, both IRF4 and ICSBP activated transcription of the IL-1beta promoter in both cell types. While this promoter is PU.1-dependent, it lacks any known PU.1/IRF composite binding sites. Synergistic activation of the IL-1beta promoter by these IRF proteins and PU.1 was found to require PU.1 serine 148. Together, these data demonstrate that IRF4 and ICSBP are dichotomous regulators of transcription in macrophages. (+info)
Kinetics of serum soluble tumour necrosis factor receptor (TNF-R) type-I and type-II after a single interferon-alpha (IFN-alpha) injection in chronic hepatitis C.
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Circulating soluble TNF receptors, which act as TNF inhibitors, increase following the administration of IFN-alpha. Whether this is due to a direct IFN action or to indirect mechanisms involving the release of other cytokines is unclear. The kinetics of serum IFN, TNF, IL-6, IL-10, soluble TNF receptor type-I (sTNF-RI) and sTNF-RII were evaluated by enzyme immunoassays in 11 patients with chronic hepatitis C, following the first dose of recombinant human IFN-alpha2b (3 MU given subcutaneously). sTNF-RI concentrations paralleled IFN concentrations, rising from a mean +/- s.e.m. value of 3.5 +/- 0.3 ng/ml at baseline to a peak value of 5.5 +/- 0.5 ng/ml after 9 h, followed by a return to 4.1 +/- 0.4 ng/ml after 24 h (P = 0.0001). sTNF-RII concentrations, which were 7.6 +/- 0.5 ng/ml at baseline, fell initially to 6.9 +/- 0.5 ng/ml, to reach a peak at 24 h of 9.0 +/- 0.7 ng/ml (P < 0.0001). In contrast, the concentrations of TNF, IL-6 and IL-10 fluctuated with no significant changes at any time point. The area under the curve (AUC) of incremental IFN values had a strong positive correlation with the AUC of incremental sTNF-RI values (r = 0.75, P < 0.01). In patients with hepatitis C, IFN concentrations reached after a single dose of IFN were paralleled by correlationally increased concentrations of sTNF-RI, which are a much better marker of administered IFN than sTNF-RII, IL-6 or IL-10. (+info)
Replication of GB virus C (hepatitis G virus) in interferon-resistant Daudi cells.
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We previously reported that Daudi cells, a Burkitt's lymphoma cell line, were capable of supporting productive infection of hepatitis C virus (HCV). During continual cultivation after HCV infection, the culture became resistant to interferons (IFNs). This resistant cell line, coded as H-903, was used as host cells for replication of GB virus C (GBV-C), also known as hepatitis G virus. GBV-C RNA was detected in the culture by reverse transcription-PCR for more than 130 days after inoculation, while it was detected for 44 days but not later in the parental IFN-sensitive Daudi cells. Productive infection of GBV-C in the H-903 system was confirmed by serially inoculating supernatants from infected cultures into uninfected cells. The viral E2 antigen was detected by immunofluorescence in the cells inoculated with the fifth passage of GBV-C. The presumed capsid-coding region of the viral genome in the inoculum, in the serially passaged virus, or in the virus produced by a long-term culture was only 16 amino acids long, suggesting that the GBV-C with a short core sequence was replication competent. (+info)
Interferons inhibit activation of STAT6 by interleukin 4 in human monocytes by inducing SOCS-1 gene expression.
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Interferons (IFNs) inhibit induction by IL-4 of multiple genes in human monocytes. However, the mechanism by which IFNs mediate this inhibition has not been defined. IL-4 activates gene expression by inducing tyrosine phosphorylation, homodimerization, and nuclear translocation of the latent transcription factor, STAT6 (signal transducer and activator of transcription-6). STAT6-responsive elements are characteristically present in the promoters of IL-4-inducible genes. Because STAT6 activation is essential for IL-4-induced gene expression, we examined the ability of type I and type II IFNs to regulate activation of STAT6 by IL-4 in primary human monocytes. Pretreatment of monocytes with IFN-beta or IFN-gamma, but not IL-1, IL-2, macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, IL-6, or transforming growth factor beta suppressed activation of STAT6 by IL-4. This inhibition was associated with decreased tyrosine phosphorylation and nuclear translocation of STAT6 and was not evident unless the cells were preincubated with IFN for at least 1 hr before IL-4 stimulation. Furthermore, inhibition by IFN could be blocked by cotreatment with actinomycin D and correlated temporally with induction of the JAK/STAT inhibitory gene, SOCS-1. Forced expression of SOCS-1 in a macrophage cell line, RAW264, markedly suppressed trans-activation of an IL-4-inducible reporter as well as IL-6- and IFN-gamma-induced reporter gene activity. These findings demonstrate that IFNs inhibit IL-4-induced activation of STAT6 and STAT6-dependent gene expression, at least in part, by inducing expression of SOCS-1. (+info)
Interferon gene transfer by a hepatitis B virus vector efficiently suppresses wild-type virus infection.
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Hepatitis B viruses specifically target the liver, where they efficiently infect quiescent hepatocytes. Here we show that human and avian hepatitis B viruses can be converted into vectors for liver-directed gene transfer. These vectors allow hepatocyte-specific expression of a green fluorescent protein in vitro and in vivo. Moreover, when used to transduce a type I interferon gene, expression of interferon efficiently suppresses wild-type virus replication in the duck model of hepatitis B virus infection. These data suggest local cytokine production after hepatitis-B-virus-mediated gene transfer as a promising concept for the treatment of acquired liver diseases, including chronic hepatitis B. (+info)
2'-adenylated derivatives of Ap3A activate RNase L.
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The exact physiological function of Ap3A (A5'ppp5"A, 5'5" diadenosine triphosphate) remains unclear. Previously we have demonstrated that the human p46 2-5A synthetase (OAS1) efficiently utilises Ap3A as an acceptor substrate for oligoadenylate synthesis. Here we show that Ap3A(2'p5'A)n oligonucleotides can activate the 2-5A-dependent RNase (RNase L), when the number of 2',5'-linked adenyl residues is two or more. Under the experimental conditions applied the half-maximal activation (AC50) of RNase L for 2'-adenylated Ap3A derivatives was determined to be in nanomolar range while the AC50 for 2-5A3 was 0.4 nM. The Ap3A(2'p5'A)n oligonucleotides are thus less effective in activating RNase L than 2-5A. We also investigated the occurrence of 2'-adenylated Ap3A in interferon and poly(I).poly(C)-treated HeLa cells. In purified trichloroacetic acid-soluble extracts about 40% of RNase L-activating material is resistant to phosphatase treatment, whereas the removal of 5'-terminal phosphates greatly reduces the activating properties of 2-5A. We assume that this activity at least partly may be associated with the presence of 2'-adenylated ApnA derivatives with blocked 5'-terminal phosphates. (+info)
Three unrelated viral transforming proteins (vIRF, EBNA2, and E1A) induce the MYC oncogene through the interferon-responsive PRF element by using different transcription coadaptors.
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Kaposi sarcoma-associated herpesvirus vIRF is a viral transcription factor that inhibits interferon signaling and transforms NIH 3T3 cells, but does not bind interferon-stimulated response element (ISRE) DNA sequences. Here we show that induction of the MYC protooncogene is required for cell transformation by vIRF, and that vIRF increases MYC transcription up to 15-fold through specific promoter interactions at an ISRE sequence called the plasmacytoma repressor factor (PRF) element. These effects are resistant to cycloheximide but are inhibited by a dominant-negative ISRE-binding protein, indicating that vIRF acts together with a cellular cofactor at the PRF element to directly transactivate MYC. The coadaptor CREB-binding protein (CBP) binds vIRF and synergizes transactivation of MYC, but, unexpectedly, closely related histone acetyltransferases p300 and P/CAF potently suppress vIRF transactivation. On the basis of the prediction that other interferon-inhibiting viral transforming proteins behave similarly, we found that Epstein-Barr virus-induced nuclear antigen 2 (EBNA2) also binds p300/CBP, and that both EBNA2 and adenovirus E1A transactivate MYC through the PRF element. For E1A, P/CAF coactivates MYC, whereas both p300 and CBP suppress E1A transactivation. For EBNA2, both P/CAF and CBP coactivate the MYC promoter, whereas p300 suppresses EBNA2 transactivation. These findings demonstrate that viral transforming proteins can activate as well as inhibit transcription through coadaptor interactions. At some promoters CBP and p300 have previously unrecognized, competitive antagonism to each other. While all three viral proteins target the same promoter element, each has a different coadaptor use profile. These findings are consistent with cellular MYC repression playing a role in innate immunity as well as in control of cell proliferation. (+info)