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.