RACK1 mediates rewiring of intracellular networks induced by hepatitis C virus infection.

Hepatitis C virus (HCV) is a positive-strand RNA virus replicating in a membranous replication organelle composed primarily of double-membrane vesicles (DMVs) having morphological resemblance to autophagosomes. To define the mechanism of DMV formation and the possible link to autophagy, we conducted a yeast two-hybrid screening revealing 32 cellular proteins potentially interacting with HCV proteins. Among these was the Receptor for Activated Protein C Kinase 1 (RACK1), a scaffolding protein involved in many cellular processes, including autophagy. Depletion of RACK1 strongly inhibits HCV RNA replication without affecting HCV internal ribosome entry site (IRES) activity. RACK1 is required for the rewiring of subcellular membranous structures and for the induction of autophagy. RACK1 binds to HCV nonstructural protein 5A (NS5A), which induces DMV formation. NS5A interacts with ATG14L in a RACK1 dependent manner, and with the ATG14L-Beclin1-Vps34-Vps15 complex that is required for autophagosome formation. Both RACK1 and ATG14L are required for HCV DMV formation and viral RNA replication. These results indicate that NS5A participates in the formation of the HCV replication organelle through interactions with RACK1 and ATG14L.

New potent biaryl sulfate-based hepatitis C virus inhibitors.

The discovery of a new series of potent hepatitis C virus (HCV) NS5A inhibitors containing biaryl sulfone or sulfate cores is reported. Structure-activity relationship (SAR) studies on inhibitors containing various substitution patterns of the sulfate or sulfone core structure established that m-,m'- substituted biaryl sulfate core-based inhibitors containing an amide moiety (compound 20) or an imidazole moiety (compound 24) showed extremely high potency. Compound 20 demonstrated double-digit pM potencies against both genotype 1b (GT-1b) and 2a (GT-2a). Compound 24 also exhibited double-digit pM potencies against GT-1b and sub nM potencies against GT-2a. Furthermore, compounds 20 and 24 exhibited no cardiotoxicity in an hERG ligand binding assay and showed acceptable plasma stability and no mutagenic potential in the Ames test. In addition, these compounds showed distinctive additive effects in combination treatment with the NS5B targeting drug sofosbuvir (Sovaldi®). The results of this study showed that the compounds 20 and 24 could be effective HCV inhibitors.

New Mechanism of Hepatic Fibrogenesis: Hepatitis C Virus Infection Induces Transforming Growth Factor ß1 Production through Glucose-Regulated Protein 94.

UNLABELLED: Hepatitis C virus (HCV) is one of the leading causes of chronic liver inflammatory disease (hepatitis), which often leads to more severe diseases, such as liver fibrosis, cirrhosis, and hepatocellular carcinoma. Liver fibrosis, in particular, is a major pathogenic consequence of HCV infection, and transforming growth factor ß1 (TGF-ß1) plays a key role in its pathogenesis. Several HCV proteins have been suggested to either augment or suppress the expression of TGF-ß1 by HCV-infected cells. Here, we report that TGF-ß1 levels are elevated in HCV-infected hepatocytes cultured in vitro and in liver tissue of HCV patients. Notably, the level of TGF-ß1 in media from in vitro-cultured HCV-infected hepatocytes was high enough to activate primary hepatic stellate cells isolated from rats. This indicates that TGF-ß1 secreted by HCV-infected hepatocytes is likely to play a key role in the liver fibrosis observed in HCV patients. Moreover, we showed that HCV E2 protein triggers the production of TGF-ß1 by HCV-infected cells through overproduction of glucose-regulated protein 94 (GRP94). IMPORTANCE: Hepatic fibrosis is a critical step in liver cirrhosis caused by hepatitis C virus infection. It is already known that immune cells, including Kupffer cells, mediate liver fibrosis. Recently, several papers have suggested that HCV-infected hepatocytes also significantly produce TGF-ß1. Here, we provide the first examination of TGF-ß1 levels in the hepatocytes of HCV patients. Using an HCV culture system, we showed that HCV infection increases TGF-ß1 production in hepatocytes. Furthermore, we confirmed that the amount of TGF-ß1 secreted by HCV-infected hepatocytes was sufficient to activate primary hepatic stellate cells. To understand the molecular basis of TGF-ß1 production in HCV-infected hepatocytes, we used HCV replicons and various stable cell lines. Finally, we elucidated that HCV E2 triggered TGF-ß1 secretion via GRP94 mediated NF-κB activation. This study contributes to the understanding of liver fibrosis by HCV and suggests a new potential target (GRP94) for blocking liver cirrhosis in HCV patients.

Infectivity of hepatitis C virus correlates with the amount of envelope protein E2: development of a new aptamer-based assay system suitable for measuring the infectious titer of HCV.

Various forms of hepatitis C virus (HCV)-related particles are produced from HCV-infected cells. Measuring infectivity of a HCV sample with the conventional 'foci counting method' is laborious and time-consuming. Moreover, the infectivity of a HCV sample does not correlate with the amount of viral RNA that can be measured by real-time RT-PCR. Here we report a new assay suitable for quantifying infectious HCV particles using aptamers against HCV E2, which is named 'Enzyme Linked Apto-Sorbent Assay (ELASA)'. The readout value of HCV ELASA linearly correlates with the infectious dose of an HCV sample, but not with the amount of HCV RNA. We also demonstrated that the activities of anti-HCV drugs can be monitored by HCV ELASA. Therefore, HCV ELASA is a quick-and-easy method to quantify infectious units of HCV stocks and to monitor efficacies of potential anti-HCV drugs.

Hepatitis C virus infection is blocked by HMGB1 released from virus-infected cells.

High-mobility group box 1 (HMGB1), an abundant nuclear protein that triggers host immune responses, is an endogenous danger signal involved in the pathogenesis of various infectious agents. However, its role in hepatitis C virus (HCV) infection is not known. Here, we show that HMGB1 protein is translocated from the nucleus to cytoplasm and subsequently is released into the extracellular milieu by HCV infection. Secreted HMGB1 triggers antiviral responses and blocks HCV infection, a mechanism that may limit HCV propagation in HCV patients. Secreted HMGB1 also may have a role in liver cirrhosis, which is a common comorbidity in HCV patients. Further investigations into the roles of HMGB1 in the diseases caused by HCV infection will shed light on and potentially help prevent these serious and prevalent HCV-related diseases.