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Hepatitis C virus (HCV) is a major cause of liver disease but the full impact of HCV infection on the hepatocyte is poorly understood. RNA sequencing (RNA-Seq) is a novel method to analyze the full transcriptional activity of a cell or tissue, thus allowing new insight into the impact of HCV infection. We conducted the first full-genome RNA-Seq analysis in a host cell to analyze infected and noninfected cells, and compared this to microarray and proteomic analyses. The combined power of the triple approach revealed that HCV infection affects a number of previously unreported canonical pathways and biological functions, including pregnane X receptor/retinoic acid receptor activation as a potential host antiviral response, and integrin-linked kinase signaling as an entry factor. This approach also identified several mechanisms implicated in HCV pathogenesis, including an increase in reactive oxygen species. HCV infection had a broad effect on cellular metabolism, leading to increases in cellular cholesterol and free fatty acid levels, associated with a profound and specific decrease in cellular glucose levels. Conclusion: RNA-Seq technology, especially when combined with established methods, demonstrated that HCV infection has potentially wide-ranging effects on cellular gene and protein expression. This in vitro study indicates a substantial metabolic impact of HCV infection and highlights new mechanisms of virus–host interaction which may be highly relevant to pathogenesis in vivo. (Hepatology 2010;52:443–453)

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Claudins regulate paracellular permeability in different tissues. The claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) is a known modulator of a claudin subset. However, it does not efficiently bind to claudin-1 (Cldn1). Cldn1 is a pharmacological target since it is (i) an essential co-receptor for hepatitis C virus (HCV) infections and (ii) a key element of the epidermal barrier limiting drug delivery. In this study, we investigated the potential of a Cldn1-binding cCPE mutant (i) to inhibit HCV entry into hepatocytes and (ii) to open the epidermal barrier. Inhibition of HCV infection by blocking of Cldn1 with cCPE variants was analyzed in the Huh7.5 hepatoma cell line. A model of reconstructed human epidermis was used to investigate modulation of the epidermal barrier by cCPE variants. In contrast to cCPEwt, the Cldn1-binding cCPE-S305P/S307R/S313H inhibited infection of Huh7.5 cells with HCV in a dose-dependent manner. In addition, TJ modulation by cCPE variant-mediated targeting of Cldn1 and Cldn4 opened the epidermal barrier in reconstructed human epidermis. cCPE variants are potent claudin modulators. They can be applied for mechanistic in vitro studies and might also be used as biologics for therapeutic claudin targeting including HCV treatment (host-targeting antivirals) and improvement of drug delivery.

We evaluated interferon (IFN) and ribavirin (RBV) as dual therapy and as part of triple-combination therapies with the iminosugars N-butyl-deoxynojirimycin (NB-DNJ), N-nonyl-deoxynojirimycin, and N-7-oxanonyl-6-deoxymethyl-galactonojirimycin. The ability of these compounds to clear bovine viral diarrhea virus (BVDV), a surrogate model for hepatitis C virus (HCV), from a persistently infected Madin-Darby bovine kidney cells cell line was determined by monitoring the secretion of viral RNA and the infectivity of secreted virions. In the BVDV system, after treatment with IFN-RBV alone, viral rebound was observed immediately after removal of the drugs. In contrast, we demonstrate that a triple drug combination of IFN, RBV, and an iminosugar eradicated the BVDV infection in a time-and a dose-dependent manner, leading to sustained viral clearance. Importantly, in the case of NB-DNJ, the sustained viral clearance was achieved by using physiologically relevant and tolerated drug concentrations. Therefore, the use of a triple-combination therapy that includes an iminosugar may prove to be of greater therapeutic value for the treatment of HCV infection than the use of IFN-RBV alone.Chronic hepatitis in humans is often caused by persistent infection with hepatitis C virus (HCV). This persistent infection commonly leads to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Pegylated alpha interferon (IFN) in combination with ribavirin (RBV) is the current treatment of choice for HCV infection (15). However, the treatment outcome is viral genotype specific, and the treatment is not effective in up to 50% of cases (8). Additional therapies aimed at the total and permanent eradication of the virus from patients are urgently required.Iminosugar derivatives are a group of compounds that have been evaluated as potential antivirals by use of the bovine viral diarrhea virus (BVDV) system (2,6,7,28). Iminosugars exert their antiviral activity by one or more modes of action, depending on the sugar analogue head group and the length of the alkyl side chain that they carry. Deoxynojirimycin (DNJ)-based iminosugar derivatives exert their antiviral effects by impairing the correct folding of the viral envelope glycoproteins of BVDV (2, 6) and other enveloped viruses, including hepatitis B virus (25), human immunodeficiency virus (12), and HCV (3), via inhibition of the host-cell encoded endoplasmic reticulum (ER) ␣-glucosidases I and II, which prevents the crucial interaction of the viral envelope glycoproteins with the cellular chaperones calnexin and calreticulin. Treatment with DNJbased iminosugars causes an inhibition of HCV E1-E2 assembly and the incorporation into HCV pseudoparticles, as well as a reduction in viral infectivity due to the incorporation of some misfolded glycoprotein complexes into virions (3). A second antiviral mechanism of action can be attributed to the alkyl chains attached to the sugar analogue head group of iminosugars. Long alkyl side chains attached to the head group are able to inhibit the HCV p7 ion...