Hepatitis C virus (HCV) is a major etiological agent of chronic liver disease and hepatocellular carcinoma (HCC). We demonstrate herewith that HCV core proteins encoded by sequences isolated from HCC tumor tissues, but not those derived from their non-tumor counterparts in the same liver, co-localise in vitro and in vivo and co-immunoprecipitate with PKR in hepatocytic Huh7 cells. We show that this association in fact augments the autophosphorylation of PKR and the phosphorylation of the translation initiation factor eIF2a, which are two markers of PKR activity. The present study therefore identi®es a novel model of viruscell interactions whereby a viral protein, the HCV core, activates PKR activity. Oncogene (2001) 20, 5836 ± 5845.

The impact of hepatitis C virus NS5A protein mutations on interferon alfa (IFN-␣) signaling pathway, cell proliferation, and viability is an important issue that is still under debate. We have therefore combined transient and stable expression in a human hepatocytic cell line (Huh7) of 3 full-length NS5A sequences, isolated from patients with or without response to IFN-␣ therapy. Expression of all 3 NS5A-reduced IFN-␣ global antiviral activity on both vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) replication. We did not show, however, an effect of these 3 NS5A proteins on double-stranded RNA-dependent kinase (PKR) expression and activity as well as colocalization and coimmunoprecipitation between NS5A and PKR. We also failed to show an effect of the 3 NS5A mutants tested on cell proliferation and viability. Overall Hepatitis C virus (HCV) is a positive-stranded RNA virus classified as hepacivirus in the Flaviridae family, which exhibits marked viral heterogeneity. 1 The polyprotein precursor is co-and post-translationally processed by both cellular and viral proteases to yield mature, structural and nonstructural proteins. 2-4 HCV is a highly prevalent pathogen and at least 300 million individuals are chronically infected worldwide. 5 The establishment of chronic infection is a major feature of HCV infection, because around 70% to 80% of acutely infected subjects will become chronic carriers, with a high subsequent risk of progression to cirrhosis and hepatocarcinoma. 6 Therapy using interferon alfa (IFN-␣) alone, or in combination with ribavirin, has been shown to be effective in chronic hepatitis C infection, but only 8% to 12% and 30% to 40% of treated patients, respectively, depending on HCV type, viral load, and quasispecies complexity, show a sustained virologic response. [7][8][9] In this context, appraisal of the mechanisms of viral resistance to IFN-␣ therapy is crucial.Several studies have suggested a direct interplay between HCV and IFN-␣-induced cellular signaling. 10,11 Expression of HCV polyprotein in osteosarcoma cells inhibits Jak1-STAT signaling. 12 HCV nonstructural protein NS5A of HCV type 1 has been particularly investigated. In Japanese HCV-1b isolates, an inverse correlation was indeed observed between the number of mutations in a region of NS5A, referred to as the interferon-sensitivity determining region (ISDR; position 2209-2248), and the response to IFN-␣ treatment. 13,14 Such a strict correlation between ISDR mutations and treatment efficacy has not, however, been shown in most studies performed in Europe and the United States. [15][16][17][18][19][20][21] Furthermore, NS5A sequence variability in domains located outside the ISDR, and in particular in the C-terminal part of the protein, is probably also implicated in therapeutic efficacy. 22 Expression of fulllength NS5A-1b but not of ISDR-deleted mutant, renders osteosarcoma 11 and murin fibroblastic cell lines 23 partially resistant to the antiviral effect of IFN-␣ against encephalomyocarditis virus (EMCV) an...

Human interferons‐alpha, ‐beta and ‐gamma enhance HLA‐DR mRNAs in all the human lymphoblastoid and melanoma cell lines studied. The increase concerns both alpha and beta chain mRNAs. Moreover, we show that immune interferon‐gamma preferentially enhances class II MHC mRNA. This effect of IFN‐gamma on the synthesis of alpha and beta HLA‐DR chains has been also analysed by immunoprecipitation. It is abolished by a monoclonal antibody directed against human IFN‐gamma. The effect of interferon on the cell surface level of HLA‐DR molecules does not always correspond to the enhancement of HLA‐DR mRNA. Our experiments suggest that this discrepancy between the enhancement of HLA‐DR mRNA and cell surface antigen might be due to a constitutively high level of the corresponding antigens on several of the human cells studied.

We identified a candidate Interferon-Stimulated Response Element (ISRE), overlapping with an Interferon Regulatory Factors binding motif (IRF-E) in the promoter region of SMN and SMNc genes. Both ISRE and IRF-E motifs are involved in mediating transcriptional induction of interferon-stimulated gene expression. We, therefore, investigated whether SMN and SMNc genes were regulated by interferons (IFN). Here we show that both IFN-beta and IFN-gamma rapidly induced SMN and SMNc mRNA and protein expression in various cell lines. The transcription factor IRF-1 bound to the candidate ISRE/IRF-E sequence of SMN and SMNc genes in vitro and overexpression of IRF-1 induced expression of both genes in transfection assays. IRF-1 is, therefore, at least in part responsible for the induction of SMN and SMNc by IFNs. In primary culture of fibroblasts from SMA patients, IFN-beta and IFN-gamma induced SMNc gene expression and restored protein defect.

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In human cells treated with interferon, there is an increase in the amount of HLA-A, B, C mRNA and, to a lower extent, membrane-bound antigen. However, the mechanism of this mRNA enhancement is still unknown. Using mouse L cells transfected with a unique class I HLA gene, we were able to show that both the related HLA mRNA and protein are increased after murine but not human interferon treatment. Moreover, the discrepancy between interferon-directed HLA mRNA and protein enhancement is also observed. The mouse transfected cells allowed us to study more precisely the origin of this discrepancy.

Ewing's sarcoma is the second most common human bone tumor in childhood. Here, we investigated the sensitivity of the Ewing tumor cell line, SK-N-MC, to the apoptotic effect of type I (IFNa) and type II (IFNc) interferons and TNFa. We demonstrate that although IFNa and TNFa alone are unable to induce cell death, they act in synergy with IFNc to induce SK-N-MC cell apoptosis. The synergistic induction of apoptosis correlated with the synergistic induction of TNFa-related apoptosis-inducing ligand (TRAIL) mRNA and TRAIL protein synthesis as well as of TRAIL secretion. Preparations of inducer-free supernatants from SK-N-MC cells stimulated with combinations of cytokines were shown to be cytotoxic for untreated SK-N-MC cells. This cytotoxicity was partially inhibited by addition of TRAILR2/Fc fusion protein, indicating that the secreted TRAIL mediates, at least in part, the apoptotic effect displayed by the supernatants of stimulated SK-N-MC cells. We have shown that the presence of IFNc is required to allow the sustained expression of IRF1 in SK-N-MC cells stimulated by addition of IFNa or TNFa suggesting that IRF1 plays a role in the synergistic induction of apoptosis by combinations of cytokines. Furthermore, we have shown that inhibition of NF-jB activation contributes to the IFNc-mediated sensitization to the apoptotic effect of TNFa. To our knowledge, this is the first report showing that interferon/cytokine combinations are able to induce TRAIL gene expression and TRAIL protein synthesis and secretion in Ewing sarcoma-derived cells. We believe that the observations reported here might contribute to the development of alternative new approaches to the treatment of Ewing tumors resistant to conventional therapy.

The β  2-adrenoceptor agonists salbutamol and fenoterol were tested for their regulatory effects on human monocyte phenotype and functions, either alone or in combination with interleukin-4 (IL-4). These drugs enhanced in a dose-dependent manner the IL-4-induced membrane and mRNA expression of the low-affinity receptor for immunoglobulin E (IgE) (CD23), as well as the release of its soluble form, sCD23. Salbutamol and fenoterol alone elicited expression of the monomorphic β  2-chain (CD18) of the leukocyte functional antigen (LEA1) family. This effect appeared to be restricted to CD11b (CR3) and CD11c (gp 150–95), because CD11a (LFA-1α chain) was not modified, β  2-Adrenoceptor stimulation was also found to potentiate the effect of IL-4 on CD11b, CD11c, and CD18 expression. In contrast, these agents alone did not alter the level of major histocompatibility complex class II and CD14 antigens or modify their respective up- and down-regulation by IL-4. Ligation of CD23 on IL-4-preincubated (CD23*) monocytes with IgE/anti-IgE immune complexes induced the release of free radicals nitric oxide and of the proinflammatory mediators IL-6 and thromboxane B2 (TxB2). Addition of salbutamol, inactive alone, potentiated the generation of superoxide anion and of nitric oxide generation, as well as the production of IL-6 and TxB2 triggered by CD23 ligation. These results indicate that β  2-adrenoceptor stimulation potentiates in vitro the IL-4-induced phenotypical and functional changes on monocytes and suggest that such an interaction could occur in IgE-dependent immune reactions. J. Leukoc. Biol. 55: 313–320; 1994.