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Background Hepatitis C virus (HCV) proteins interfere with the interferon (IFN)-alpha-induced Jak/signal transducer and activator of transcription (STAT) pathway. Which protein is responsible for this effect and whether this interference results in down-regulation of IFN-induced genes remain controversial. We analyzed the effect of HCV core (HCV-Co) protein on expression of IFN-induced antiviral genes. Methods HepG2 cells were transfected with the plasmid pHCV-Co, and, after treatment with IFN-alpha , levels of MxA, protein kinase R (PKR), and 2'-5' oligoadenylate synthetase (2'-5'OAS) mRNA were determined. Chloramphenycol acethyl transferase (CAT) analysis was performed on cells cotransfected with pHCV-Co and pMx4CAT (containing the MxA gene promoter) and treated with IFN. Electrophoretic mobility shift assays were used, and Western-blot analysis of STAT 1 and 2 was performed. Results Levels of MxA mRNA in pHCV-Co-transfected cells decreased in a dose-dependent manner, by down-regulation of the MxA gene promoter. HCV-Co protein inhibits binding of IFN-stimulated gene factor 3 (ISGF3) to the IFN-stimulated response element (ISRE). Intracellular distribution of STAT 1 and 2 was not modified after treatment with IFN. Expression of HCV-Co protein also results in down-regulation of expression of PKR and 2'-5'OAS genes. Conclusion HCV-Co protein inhibits IFN-alpha-induced transcription of antiviral genes by decreasing binding of ISGF3 to the ISRE.

Chronic hepatitis C in children is characterized by milder forms of liver damage than those found in adults. Such a difference has been attributed to a low viral load in children that may lead to poor recognition of infected cells by the immune system. One approach that could be used to confirm this hypothesis may be to examine the number of infected hepatocytes in liver biopsies. Paraffin embedded liver biopsies from 21 children and 15 adults with chronic hepatitis C virus (HCV) infection (with a similar duration of the infection) were hybridized in situ and the percentage of infected hepatocytes was correlated with the histological activity index, alanine aminotransferase levels and HCV viraemia levels. Histological activity index and HCV viraemia levels were statistically higher (P

Intrahepatic levels of the inducible nitric oxide synthase (iNOS) are increased in chronic hepatitis C patients. As iNOS gene promoter contains Nuclear Factor (NF)-kappaB binding sites and hepatitis C virus (HCV) core protein activates NF-kappaB, the aim of this work was to study if HCV core protein transactivates iNOS promoter through NF-kappaB activation. iNOS mRNA and protein were determined by RT-PCR and western blot in HepG2 cells. The effect of HCV core protein on iNOS promoter was assayed by cotransfecting HepG2 cells with the core protein expression plasmid pHCV-Co and p1iNOS-CAT or p2iNOS-CAT plasmids. Formation of NF-kappaB-DNA complexes was determined by electrophoretic mobility shift assay. Transfection of HepG2 cells with pHCV-Co plasmid results in an increase in iNOS mRNA and protein levels. Cotransfection with pHCV-Co and p1iNOS-CAT or p2iNOS-CAT plasmids results in a transactivation of iNOS promoter, the presence of the proximal NF-kappaB binding site in the promoter being sufficient for the transactivation. Furthermore, the HCV core protein increases the formation of complexes between NF-kappaB and its binding sequence in the iNOS promoter. The expression of the NF-kappaB inhibitor IKB reverts the effect of the HCV core protein on the iNOS promoter. In conclusion, HCV core protein transactivates iNOS gene promoter through NF-kappaB activation.

Background There are patients in whom the etiology of long-standing abnormal results of liver-function tests is unknown (ALF-EU) after exclusion of all known causes of liver diseases. We analyzed the presence of hepatitis C virus (HCV) RNA in liver-biopsy specimens from 100 patients who were negative for anti-HCV antibodies and for serum HCV RNA and who had ALF-EU. Methods HCV RNA status was tested by reverse-transcription polymerase chain reaction (RT-PCR) and by in situ hybridization, in liver and peripheral-blood mononuclear cells (PBMCs). Results HCV RNA was detected in liver-biopsy specimens from 57 of 100 patients negative for anti-HCV antibodies and for serum HCV RNA (i.e., who had occult HCV infection). HCV RNA of negative polarity was found in the liver of 48 (84.2%) of these 57 patients with occult HCV infection. Nucleotide-sequence analysis confirmed the specificity of detection of HCV RNA and that patients were infected with the HCV 1b genotype. Of these 57 patients with intrahepatic HCV RNA, 40 (70%) had viral RNA in their PBMCs. With regard to liver histology, patients with occult HCV infection were more likely to have necroinflammatory activity (P=.017) and fibrosis (P=.022) than were patients without intrahepatic HCV RNA. Conclusions Patients with ALF-EU may have intrahepatic HCV RNA in the absence of anti-HCV antibodies and of serum HCV RNA.

It has not been completely elucidated whether the liver injury induced by the hepatitis C virus (HCV) is due to direct cytopathic damage or to an immune-mediated response against HCV-infected hepatocytes. In this work, we have determined the percentage of HCV-infected hepatocytes, the histological activity index, and the viremia levels in chronically HCV-infected patients with different grades of liver injury to investigate any possible correlation between them. For that purpose, liver biopsies from 27 patients with HCV chronic hepatitis were analyzed by in situ hybridization. This technique revealed that the percentage of infected hepatocytes ranged from 0.04% to 83.6%. Regarding the viremia levels, HCV RNA concentration ranged from 1.8 × 10 3 to 1.4 × 10 6 genome copies/ml. A significant correlation ( r = 0.54; P = 0.003) between the percentage of infected hepatocytes and the viremia levels was found. In contrast, no correlation was observed between the percentage of HCV-infected hepatocytes or the viremia levels and the histological activity index. In conclusion, we have shown that the HCV viremia reflects the extent of the infection in the liver and that the liver injury in chronic HCV infection is not directly related to either the number of infected hepatocytes or the serum HCV RNA concentration.