Hepatitis C virus (HCV) is a major risk factor for human hepatocellular carcinoma (HCC) but the mechanisms underlying HCV-induced carcinogenesis are still poorly understood. We have hypothesized that viral variants, selected during long-term infection, might contribute to cellular transformation. To address this issue, we have investigated the effect of natural HCV core variants isolated from liver tumors (T), or their non-tumor (NT) counterparts, on the tumor growth factor-b (TGF-b) pathway, a major regulator of cellular proliferation, differentiation and apoptosis. We have found a significant reduction in TGF-b reporter gene activity with the expression of core sequences isolated from liver tumors. In contrast, moderate or no effects were observed with non-tumor mutants or a core reference sequence. The molecular mechanisms have been characterized and involved the inhibition, by tumor-derived cores, of the DNA-binding activity of the Smad3/4 transcription factors complex. This inhibition occurs through a direct interaction between the central domain (amino acids 59-126) of tumor-derived core and the MH1 DNA-binding domain of Smad3, thus preventing its binding to DNA. We have therefore identified a new cell-signaling pathway targeted by HCV core and inhibited by tumor-derived core sequences. These results suggest that during chronic infection, there is selection of viral variants that may promote cell transformation by providing, to clonally expanding cells, resistance to TGF-b antiproliferative effects. Oncogene (2005) 24, 6119-6132.

Deglycosylation of viral glycoproteins has been shown to influence the number of available epitopes and to modulate immune recognition of antigens. We investigated the role played by N-glycans in the immunogenicity of hepatitis C virus (HCV) E1 envelope glycoprotein, a naturally poor immunogen. Eight plasmids were engineered, encoding E1 protein mutants in which the four N-linked glycosylation sites of the protein were mutated separately or in combination. In vitro expression studies showed an influence of N-linked glycosylation on expression efficiency, instability, and/or secretion of the mutated proteins. Immunogenicity of the E1 mutants was studied in BALB/c mice following intramuscular and intraepidermal injection of the plasmids. Whereas some mutations had no or only minor effects on the antibody titers induced, mutation of the fourth glycosylation site (N4) significantly enhanced the anti-E1 humoral response in terms of both seroconversion rates and antibody titers. Moreover, antibody induced by the N4 mutant was able to recognize HCV-like particles with higher titers than those induced by the wild-type construct. Epitope mapping indicated that the E1 mutant antigens induced antibody directed at two major domains: one, located at amino acids (aa) 313 to 332, which is known to be reactive with sera from HCV patients, and a second one, located in the N-terminal domain of E1 (aa 192 to 226). Analysis of the induced immune cellular response confirmed the induction of gamma interferon-producing cells by all mutants, albeit to different levels. These results show that N-linked glycosylation can limit the antibody response to the HCV E1 protein and reveal a potential vaccine candidate with enhanced immunogenicity.