The Hepatitis C virus NS5A and core proteins exert antagonistic effects on HAMP gene expression: the hidden interplay with the MTF-1/MRE pathway.

Hepcidin, a 25-amino acid peptide encoded by the HAMP gene and produced mainly by hepatocytes and macrophages, is a mediator of innate immunity and the central iron-regulatory hormone. Circulating hepcidin controls iron efflux by inducing degradation of the cellular iron exporter ferroportin. HCV infection is associated with hepatic iron overload and elevated serum iron, which correlate with poor antiviral responses. The HCV nonstructural NS5A protein is known to function in multiple aspects of the HCV life cycle, probably exerting its activity in concert with cellular factor(s). In this study, we attempted to delineate the effect of HCV NS5A on HAMP gene expression. We observed that transient transfection of hepatoma cell lines with HCV NS5A resulted in down-regulation of HAMP promoter activity. A similar effect was evident after transduction of Huh7 cells with a recombinant baculovirus vector expressing NS5A protein. We proceeded to construct an NS5A-expressing stable cell line, which also exhibited down-regulation of HAMP gene promoter activity and significant reduction of HAMP mRNA and hepcidin protein levels. Concurrent expression of HCV core protein, a well-characterized hepcidin inducer, revealed antagonism between those two proteins for hepcidin regulation. In attempting to identify the pathways involved in NS5A-driven reduction of hepcidin levels, we ruled out any NS5A-induced alterations in the expression of the well-known hepcidin inducers SMAD4 and STAT3. Further analysis linked the abundance of intracellular zinc ions and the deregulation of the MTF-1/MRE/hepcidin axis with the observed phenomenon. This effect could be associated with distinct phases in HCV life cycle.

Differential regulation of the Wnt/ß-catenin pathway by hepatitis C virus recombinants expressing core from various genotypes.

Clinical studies have suggested association of some hepatitis C virus (HCV) subtypes or isolates with progression toward hepatocellular carcinoma (HCC). HCV core protein has been reported to interfere with host Wnt/ß-catenin pathway, a cell fate-determining pathway, which plays a major role in HCC. Here, we investigated the impact of HCV core genetic variability in the dysregulation of Wnt/ß-catenin pathway. We used both transient expression of core proteins from clinical isolates of HCV subtypes 1a (Cambodia), 4a (Romania) and 4f (Cameroon) and infection systems based on a set of engineered intergenotypic recombinant viruses encoding core from these various clinical strains. We found that TCF transcription factor-dependent reporter activity was upregulated by core in a strain-specific manner. We documented core sequence-specific transcriptional upregulation of several ß-catenin downstream target genes associated with cell proliferation and malignant transformation, fibrogenesis or fat accumulation. The extent of ß-catenin nuclear translocation varied in accordance with ß-catenin downstream gene upregulation in infected cells. Pairwise comparisons of subgenotypic core recombinants and mutated core variants unveiled the critical role of core residues 64 and 71 in these dysregulations. In conclusion, this work identified natural core polymorphisms involved in HCV strain-specific activation of Wnt/ß-catenin pathway in relevant infection systems.