Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFN␣) and ribavirin. It achieves a sustained viral clearance in only 50 -60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFN␣. In patients with a rapid virological response to treatment, pegIFN␣ induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFN␣ did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway.Jak-STAT signaling ͉ liver ͉ viral hepatitis

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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 Gastroenterology 2019;156:2313-2329 BASIC AND TRANSLATIONAL LIVER with humanized livers. METHODS: We performed genomewide 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. RESULTS: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 ¼ 216), a subset of which (n ¼ 21) achieved viral clearance. CONCLUSIONS: 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.

A new polymorphism near the IL28B locus negatively affects induction of IL28B and exhibits strong predictive value for HCV treatment response and spontaneous resolution.

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Hepatitis C virus (HCV) entry is dependent on coreceptor complex formation between the tetraspanin superfamily member CD81 and the tight junction protein claudin-1 (CLDN1) on the host cell membrane. The receptor tyrosine kinase EGFR acts as a cofactor for HCV entry by promoting CD81-CLDN1 complex formation via unknown mechanisms. We identify the GTPase HRas, activated downstream of EGFR signaling, as a key host signal transducer for EGFR-mediated HCV entry. Proteomic analysis revealed that HRas associates with tetraspanin CD81, CLDN1, and the previously unrecognized HCV entry cofactors integrin β1 and Ras-related protein Rap2B in hepatocyte membranes. HRas signaling is required for lateral membrane diffusion of CD81, which enables tetraspanin receptor complex assembly. HRas was also found to be relevant for entry of other viruses, including influenza. Our data demonstrate that viruses exploit HRas signaling for cellular entry by compartmentalization of entry factors and receptor trafficking.

Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment-induced clearance of HCV depend on genetic variation within the interferon-lambda locus, but until now no clear causal relationship has been established. Here we demonstrate that an amino-acid substitution in the IFNl4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Patients harbouring the impaired IFNl4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared with patients coding for the fully active IFNl4-P70 variant. Altogether, these data provide evidence supporting a role for the active IFNl4 protein as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.

Recombinant alpha interferon (IFN-␣) is used for the treatment of viral hepatitis and some forms of cancer. During these therapies IFN-␣ is injected once daily or every second day for several months. Recently, the long-acting pegylated IFN-␣ (pegIFN-␣) has replaced standard IFN-␣ in therapies of chronic hepatitis C because it is more effective, supposedly by inducing a long-lasting activation of IFN signaling pathways. IFN signaling in cultured cells, however, becomes refractory within hours, and little is known about the pharmacodynamic effects of continuously high IFN-␣ serum concentrations. To investigate the behavior of the IFN system in vivo, we repeatedly injected mice with IFN-␣ and analyzed its effects in the liver. Within hours after the first injection, IFN-␣ signaling became refractory to further stimulation. The negative regulator SOCS1 was rapidly upregulated and likely responsible for early termination of IFN-␣ signaling. For long-lasting refractoriness, neither SOCS1 nor SOCS3 were instrumental. Instead, we identified the inhibitor USP18/ UBP43 as the key mediator. Our results indicate that the current therapeutic practice using long-lasting pegIFN-␣ is not well adapted to the intrinsic properties of the IFN system. Targeting USP18 expression may allow to exploit the full therapeutic potential of recombinant IFN-␣.