Hepatitis C virus (HCV) infection is a major cause of chronic liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. While searching for new natural anti-HCV agents in agricultural products, we found a potent inhibitor of HCV RNA expression in extracts of blueberry leaves when examined in an HCV subgenomic replicon cell culture system. This activity was observed in a methanol extract fraction of blueberry leaves and was purified by repeated fractionations in reversed-phase high-performance liquid chromatography. The final purified fraction showed a 63-fold increase in specific activity compared with the initial methanol extracts and was composed only of carbon, hydrogen, and oxygen. Liquid chromatography/mass-ion trap-time of flight analysis and butanolHCl hydrolysis analysis of the purified fraction revealed that the blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore, structural analysis using acid thiolysis indicated that the mean degree of polymerization of the purified proanthocyanidin was 7.7, consisting predominantly of epicatechin. Proanthocyanidin with a polymerization degree of 8 to 9 showed the greatest potency at inhibiting the expression of subgenomic HCV RNA. Purified proanthocyanidin showed dose-dependent inhibition of expression of the neomycin-resistant gene and the NS-3 protein gene in the HCV subgenome in replicon cells. While characterizing the mechanism by which proanthocyanidin inhibited HCV subgenome expression, we found that heterogeneous nuclear ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived proanthocyanidin and was indispensable for HCV subgenome expression in replicon cells. These data suggest that proanthocyanidin isolated from blueberry leaves may have potential usefulness as an anti-HCV compound by inhibiting viral replication.Hepatitis C virus (HCV) 2 is often associated with the development of chronic liver diseases. Infection by HCV causes chronic hepatitis at high rates and finally results in liver cirrhosis and subsequent occurrence of hepatocellular carcinoma (1-3). The number of people worldwide who are infected by HCV is estimated to be over 200 million with 2 million infections in Japan (4). The South Kyushu area of Japan, including Miyazaki prefecture, has a high prevalence of this virus, and it is now recognized as a social problem. There is no vaccine effective for HCV at present. The elimination of HCV may be achieved by a combination of pegylated ␣-interferon and ribavirin, a broad spectrum antiviral drug (4 -6). However, virological response to this combination therapy has been reported to be 80% for genotypes 2 and 3, but less than 50% for genotype 1 (7, 8). Moreover, ␣-interferon is associated with severe sideeffects, including leucopenia, thrombocytopenia, depression, fatigue, and flu-like symptoms, and ribavirin is associated with side-effects such as hemolytic anemia (9). Therefore, establishment of a new modality of treatment without serious adverse effects is still required.Considering the prolonged p...

SummaryHepatocellular carcinoma (HCC) is a common cancer worldwide and represents the outcome of the natural history of chronic liver disease. The growing rates of HCC may be partially attributable to increased numbers of people with non‐alcoholic fatty liver disease (NAFLD) and non‐alcoholic steatohepatitis (NASH). However, details of the liver‐specific molecular mechanisms responsible for the NAFLD–NASH–HCC progression remain unclear, and mouse models that can be used to explore the exact factors that influence the progression of NAFLD/NASH to the more chronic stages of liver disease and subsequent HCC are not yet fully established. We have previously reported a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) as a dietary NASH model with rapidly progressive liver fibrosis in mice. The current study in C57BL/6J mice fed CDAHFD provided evidence for the chronic persistence of advanced hepatic fibrosis in NASH and disease progression towards HCC in a period of 36 weeks. When mice fed CDAHFD were switched back to a standard diet, hepatic steatosis was normalized and NAFLD activity score improved, but HCC incidence increased and the phenotype of fibrosis‐associated HCC development was observed. Moreover, when mice continued to be fed CDAHFD for 60 weeks, HCC further developed without severe body weight loss or carcinogenesis in other organs. The autochthonous tumours showed a variety of histological features and architectural patterns including trabecular, pseudoglandular and solid growth. The CDAHFD mouse model might be a useful tool for studying the development of HCC from NAFLD/NASH, and potentially useful for better understanding pathological changes during hepatocarcinogenesis.

Most acute hepatitis C virus (HCV) infections become chronic and some progress to liver cirrhosis or hepatocellular carcinoma. Standard therapy involves an interferon (IFN)-α-based regimen, and efficacy of therapy has been significantly improved by the development of protease inhibitors. However, several issues remain concerning the injectable form and the side effects of IFN. Here, we report an orally available, small-molecule type I IFN receptor agonist that directly transduces the IFN signal cascade and stimulates antiviral gene expression. Like type I IFN, the small-molecule compound induces IFN-stimulated gene (ISG) expression for antiviral activity in vitro and in vivo in mice, and the ISG induction mechanism is attributed to a direct interaction between the compound and IFN-α receptor 2, a key molecule of IFN-signaling on the cell surface. Our study highlights the importance of an orally active IFN-like agent, both as a therapy for antiviral infections and as a potential IFN substitute.

The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors.

Edited by Charles E. SamuelHepatitis C virus (HCV) establishes a chronic infection that can lead to cirrhosis and hepatocellular carcinoma. The HCV life cycle is closely associated with host factors that promote or restrict viral replication, the characterization of which could help to identify potential therapeutic targets. To this end, here we performed a genome-wide microarray analysis and identified ribonucleotide reductase M2 (RRM2) as a cellular factor essential for HCV replication. We found that RRM2 is up-regulated in response to HCV infection in quiescent hepatocytes from humanized chimeric mouse livers. To elucidate the molecular basis of RRM2 expression in HCV-infected cells, we used HCV-infected hepatocytes from chimeric mice and hepatoma cells infected with the HCV strain JFH1. Both models exhibited increased RRM2 mRNA and protein expression levels. Moreover, siRNA-mediated silencing of RRM2 suppressed HCV replication and infection. Of note, RRM2 and RNA polymerase nonstructural protein 5B (NS5B) partially co-localized in cells and co-immunoprecipitated, suggesting that they might interact. RRM2 knockdown reduced NS5B expression, which depended on the protein degradation pathway, as NS5B RNA levels did not decrease and NS5B protein stability correlated with RRM2 protein levels. We also found that RRM2 silencing decreased levels of hPLIC1 (human homolog 1 of protein linking integrin-associated protein and cytoskeleton), a ubiquitin-like protein that interacts with NS5B and promotes its degradation. This finding suggests that there is a dynamic interplay between RRM2 and the NS5B-hPLIC1 complex that has an important function in HCV replication. Together, these results identify a role of host RRM2 in viral RNA replication.Hepatitis C virus (HCV) 3 is an enveloped positive-strand RNA virus belonging to the Flaviviridae family (1). HCV infects and replicates within quiescent hepatocytes, establishing a chronic infection that can lead to cirrhosis and hepatocellular carcinoma. Although effective anti-HCV drugs have been developed, their low barrier to viral resistance and inability to prevent hepatocellular carcinoma development represent major clinical challenges (2).The HCV life cycle proceeds exclusively in the cytoplasm of host cells. Upon decapsidation, the viral genome is released into the cytoplasm, where it is translated into a large polyprotein that is processed by cellular and viral proteases to yield mature structural (core, E1, and E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (3). Following translation, these proteins become associated with a membranous web derived from the endoplasmic reticulum, in which viral genome replication takes place. NS3 through NS5B constitute the replication complex, and the positive-strand RNA genome serves as a template for the RNA-dependent RNA polymerase NS5B to produce the negative-sense replicative intermediate necessary for the generation of new positive-sense RNA genomes. These are in turn used as templates for further RNA replica...