Chronic hepatitis C virus (HCV) infection is an important risk factor for hepatocellular carcinoma (HCC). Despite effective antiviral therapies, the risk for HCC is decreased but not eliminated after a sustained virologic response (SVR) to direct-acting antiviral (DAA) agents, and the risk is higher in patients with advanced fibrosis. We investigated HCV-induced epigenetic alterations that might affect risk for HCC after DAA treatment in patients and mice with humanized livers. We performed genome-wide ChIPmentation-based ChIP-Seq and RNA-seq analyses of liver tissues from 6 patients without HCV infection (controls), 18 patients with chronic HCV infection, 8 patients with chronic HCV infection cured by DAA treatment, 13 patients with chronic HCV infection cured by interferon therapy, 4 patients with chronic hepatitis B virus infection, and 7 patients with nonalcoholic steatohepatitis in Europe and Japan. HCV-induced epigenetic modifications were mapped by comparative analyses with modifications associated with other liver disease etiologies. uPA/SCID mice were engrafted with human hepatocytes to create mice with humanized livers and given injections of HCV-infected serum samples from patients; mice were given DAAs to eradicate the virus. Pathways associated with HCC risk were identified by integrative pathway analyses and validated in analyses of paired HCC tissues from 8 patients with an SVR to DAA treatment of HCV infection. We found chronic HCV infection to induce specific genome-wide changes in H3K27ac, which correlated with changes in expression of mRNAs and proteins. These changes persisted after an SVR to DAAs or interferon-based therapies. Integrative pathway analyses of liver tissues from patients and mice with humanized livers demonstrated that HCV-induced epigenetic alterations were associated with liver cancer risk. Computational analyses associated increased expression of SPHK1 with HCC risk. We validated these findings in an independent cohort of patients with HCV-related cirrhosis (n = 216), a subset of which (n = 21) achieved viral clearance. In an analysis of liver tissues from patients with and without an SVR to DAA therapy, we identified epigenetic and gene expression alterations associated with risk for HCC. These alterations might be targeted to prevent liver cancer in patients treated for HCV infection.

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide. Restriction of HCV infection to human hepatocytes suggests that liver-specific host factors play a role in the viral life cycle. Using a yeast-two-hybrid system, we identified apolipoprotein E (apoE) as a liver-derived host factor specifically interacting with HCV nonstructural protein 5A (NS5A) but not with other viral proteins. The relevance of apoE–NS5A interaction for viral infection was confirmed by co-immunoprecipitation and co-localization studies of apoE and NS5A in an infectious HCV cell culture model system. Silencing apoE expression resulted in marked inhibition of infectious particle production without affecting viral entry and replication. Analysis of particle production in liver-derived cells with silenced apoE expression showed impairment of infectious particle assembly and release. The functional relevance of the apoE–NS5A interaction for production of viral particles was supported by loss or decrease of apoE–NS5A binding in assembly-defective viral mutants. Conclusion: These results suggest that recruitment of apoE by NS5A is important for viral assembly and release of infectious viral particles. These findings have important implications for understanding the HCV life cycle and the development of novel antiviral strategies targeting HCV–lipoprotein interaction. (Hepatology 2010)

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world with an annual incidence of more than 500000 in the year 2000. Its incidence is rising in many countries. Recently, it has been estimated that about 53% of HCC cases in the world are related to hepatitis B virus (HBV). The epidemiological association of HBV with HCC is well established. In recent studies, it was revealed that HBsAg carriers have a 25-37 times increased risk of developing HCC as compared to non-infected people. At present, HBV-associated carcinogenesis can be seen as a multi-factorial process that includes both direct and indirect mechanisms that might act synergistically. The integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion. The integration has been shown in a number of cases to affect a variety of cancer-related genes and to exert insertional mutagenesis. The permanent liver inflammation, induced by the immune response, resulting in a degeneration and regeneration process confers to the accumulation of critical mutations in the host genome. In addition to this, the regulatory proteins HBx and the PreS2 activators that can be encoded by the integrate exert a tumor promoter-like function resulting in positive selection of cells producing a functional regulatory protein. Gene expression profiling and proteomic techniques may help to characterize the molecular mechanisms driving HBV-associated carcinogenesis, and thus potentially identify new strategies in diagnosis and therapy.

Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer¹. Cell entry of HCV² and other pathogens^3,4,5 is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver–chimeric mouse model⁶, we show that a monoclonal antibody specific for the TJ protein claudin-1 (ref. 7) eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection by means of host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy.