Functional microRNA screen uncovers O-linked N-acetylglucosamine transferase as a host factor modulating hepatitis C virus morphogenesis and infectivity.

OBJECTIVE: Infection of human hepatocytes by the hepatitis C virus (HCV) is a multistep process involving both viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Given that miRNAs were indicated to regulate between 30% and 75% of all human genes, we aimed to investigate the functional and regulatory role of miRNAs for the HCV life cycle. DESIGN: To systematically reveal human miRNAs affecting the HCV life cycle, we performed a two-step functional high-throughput miRNA mimic screen in Huh7.5.1 cells infected with recombinant cell culture-derived HCV. miRNA targeting was then assessed using a combination of computational and functional approaches. RESULTS: We uncovered miR-501-3p and miR-619-3p as novel modulators of HCV assembly/release. We discovered that these miRNAs regulate O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression and identified OGT and O-GlcNAcylation as regulators of HCV morphogenesis and infectivity. Furthermore, increased OGT expression in patient-derived liver tissue was associated with HCV-induced liver disease and cancer. CONCLUSION: miR-501-3p and miR-619-3p and their target OGT are previously undiscovered regulatory host factors for HCV assembly and infectivity. In addition to its effect on HCV morphogenesis, OGT may play a role in HCV-induced liver disease and hepatocarcinogenesis.

Hepatitis C virus induces a prediabetic state by directly impairing hepatic glucose metabolism in mice.

Virus-related type 2 diabetes is commonly observed in individuals infected with the hepatitis C virus (HCV); however, the underlying molecular mechanisms remain unknown. Our aim was to unravel these mechanisms using FL-N/35 transgenic mice expressing the full HCV ORF. We observed that these mice displayed glucose intolerance and insulin resistance. We also found that Glut-2 membrane expression was reduced in FL-N/35 mice and that hepatocyte glucose uptake was perturbed, partly accounting for the HCV-induced glucose intolerance in these mice. Early steps of the hepatic insulin signaling pathway, from IRS2 to PDK1 phosphorylation, were constitutively impaired in FL-N/35 primary hepatocytes via deregulation of TNFα/SOCS3. Higher hepatic glucose production was observed in the HCV mice, despite higher fasting insulinemia, concomitant with decreased expression of hepatic gluconeogenic genes. Akt kinase activity was higher in HCV mice than in WT mice, but Akt-dependent phosphorylation of the forkhead transcription factor FoxO1 at serine 256, which triggers its nuclear exclusion, was lower in HCV mouse livers. These findings indicate an uncoupling of the canonical Akt/FoxO1 pathway in HCV protein-expressing hepatocytes. Thus, the expression of HCV proteins in the liver is sufficient to induce insulin resistance by impairing insulin signaling and glucose uptake. In conclusion, we observed a complete set of events leading to a prediabetic state in HCV-transgenic mice, providing a valuable mechanistic explanation for HCV-induced diabetes in humans.

Programmed death ligand 1 expression in hepatocellular carcinoma: Relationship With clinical and pathological features.

The prognosis of hepatocellular carcinoma (HCC) remains poor, with only one third of patients eligible for curative treatments and very limited survival benefits with the use of sorafenib, the current standard of care for advanced disease. Recently, agents targeting the programmed death ligand 1 (PD-L1)/programmed death receptor 1 (PD-1) immune checkpoint were shown to display impressive antitumor activity in various solid or hematological malignancies, including HCC. PD-L1 immunohistochemical expression is thought to represent a biomarker predictive of drug sensitivity. Here, we investigated PD-L1 expression in a series of 217 HCCs and correlated our results with clinical and histological features and immunohistochemical markers (PD-1, cytokeratin 19, glutamine synthetase, and ß-catenin expression). PD-L1 expression by neoplastic cells was significantly associated with common markers of tumor aggressiveness (high serum alpha-fetoprotein levels, P = 0.038; satellite nodules, P < 0.001; macrovascular invasion, P < 0.001; microvascular invasion, P < 0.001; poor differentiation, P < 0.001) and with the progenitor subtype of HCC (cytokeratin 19 expression, P = 0.031). High PD-L1 expression by inflammatory cells from the tumor microenvironment also correlated with high serum alpha-fetoprotein levels (P < 0.001), macrovascular invasion (P = 0.001), poor differentiation (P = 0.001), high PD-1 expression (P < 0.001), and the so-called lymphoepithelioma-like histological subtype of HCC (P = 0.003). CONCLUSION: PD-L1 expression by either neoplastic or intratumoral inflammatory cells is related to tumor aggressiveness and suggests that the response to treatments targeting the PD-L1/PD-1 immune checkpoint could be restricted to particular HCC variants; thus, enrichment of these tumor subtypes in future clinical trials should be considered. (Hepatology 2016;64:2038-2046).