Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. With ∼170 million individuals infected and current interferon-based treatment having toxic side effects and marginal efficacy, more effective antivirals are crucially needed. Although HCV protease inhibitors were just approved by the US Food and Drug Administration (FDA), optimal HCV therapy, analogous to HIV therapy, will probably require a combination of antivirals targeting multiple aspects of the viral lifecycle. Viral entry represents a potential multifaceted target for antiviral intervention; however, to date, FDA-approved inhibitors of HCV cell entry are unavailable. Here we show that the cellular Niemann-Pick C1-like 1 (NPC1L1) cholesterol uptake receptor is an HCV entry factor amendable to therapeutic intervention. Specifically, NPC1L1 expression is necessary for HCV infection, as silencing or antibody-mediated blocking of NPC1L1 impairs cell culture-derived HCV (HCVcc) infection initiation. In addition, the clinically available FDA-approved NPC1L1 antagonist ezetimibe potently blocks HCV uptake in vitro via a virion cholesterol-dependent step before virion-cell membrane fusion. Moreover, ezetimibe inhibits infection by all major HCV genotypes in vitro and in vivo delays the establishment of HCV genotype 1b infection in mice with human liver grafts. Thus, we have not only identified NPC1L1 as an HCV cell entry factor but also discovered a new antiviral target and potential therapeutic agent.

/pdf/identification-of-the-niemann-pick-c1-like-1-cholesterol-26abfl8e50.pdf

Identification of the Niemann-Pick C1–like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor

Hepatitis C virus (HCV) is a leading cause of chronic liver disease, including chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatitis C infection associates with lipid and lipoprotein metabolism disorders such as hepatic steatosis, hypobetalipoproteinemia, and hypocholesterolemia. Furthermore, virus production is dependent on hepatic very-low-density lipoprotein (VLDL) assembly, and circulating virions are physically associated with lipoproteins in complexes termed lipoviral particles. Evidence has indicated several functional roles for the formation of these complexes, including co-opting of lipoprotein receptors for attachment and entry, concealing epitopes to facilitate immune escape, and hijacking host factors for HCV maturation and secretion. Here, we review the evidence surrounding pathogenesis of the hepatitis C infection regarding lipoprotein engagement, cholesterol and triglyceride regulation, and the molecular mechanisms underlying these effects.

https://www.mdpi.com/1999-4915/5/5/1292/pdf

Hepatitis C Virus, Cholesterol and Lipoproteins — Impact for the Viral Life Cycle and Pathogenesis of Liver Disease

https://www.journal-of-hepatology.eu/article/S0168-8278(14)00394-8/pdf

Strategies for immortalization of primary hepatocytes.

Dimethyl sulfoxide (DMSO) is a small molecule with polar, aprotic and amphiphilic properties. It serves as a solvent for many polar and nonpolar molecules and continues to be one of the most used solvents (vehicle) in medical applications and scientific research. To better understand the cellular effects of DMSO within the concentration range commonly used as a vehicle (0.1–1.5%, v/v) for cellular treatments, we applied Attenuated Total Reflectance (ATR) Fourier Transform Infrared (FT-IR) spectroscopy to DMSO treated and untreated epithelial colon cancer cells. Both unsupervised (Principal Component Analysis-PCA) and supervised (Linear Discriminant Analysis-LDA) pattern recognition/modelling algorithms applied to the IR data revealed total segregation and prominent differences between DMSO treated and untreated cells at whole, lipid and nucleic acid regions. Several of these data were supported by other independent techniques. Further IR data analyses of macromolecular profile indicated comprehensive alterations especially in proteins and nucleic acids. Protein secondary structure analysis showed predominance of β-sheet over α-helix in DMSO treated cells. We also observed for the first time, a reduction in nucleic acid level upon DMSO treatment accompanied by the formation of Z-DNA. Molecular docking and binding free energy studies indicated a stabilization of Z-DNA in the presence of DMSO. This alternate DNA form may be related with the specific actions of DMSO on gene expression, differentiation, and epigenetic alterations. Using analytical tools combined with molecular and cellular biology techniques, our data indicate that even at very low concentrations, DMSO induces a number of changes in all macromolecules, which may affect experimental outcomes where DMSO is used as a solvent.

Low dose dimethyl sulfoxide driven gross molecular changes have the potential to interfere with various cellular processes

In this chapter, the isolation of primary mouse hepatocytes and their response to chemical treatment are described. We show that it is important to consider, in the experimental design, the sex of the animals to be used. We demonstrate this by measuring the effect of sex hormones or xenobiotics on the expression of fl avin-containing monooxygenase 5 in cultures of primary hepatocytes isolated from male and female mice.

Protocols in In Vitro Hepatocyte Research

Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV) infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc), albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread). We found that while the early events in HCV infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56) expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection. We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection.

/pdf/permissiveness-of-human-hepatoma-cell-lines-for-hcv-24oa6emxbg.pdf

Permissiveness of human hepatoma cell lines for HCV infection

The rate of hepatitis C virus infection initiation in vitro is directly related to particle density

The objective of this study was to characterize Huh7 cells' baseline capacity to metabolize drugs and to investigate whether the drug metabolism was enhanced upon treatment with dimethyl sulfoxide (DMSO). The messenger RNA (mRNA) levels of major Phase I and Phase II enzymes were determined by quantitative real-time-polymerase chain reaction (RT-PCR), and activities of major drug-metabolizing enzymes were examined using probe drugs by analysing relevant metabolite production rates. The expression levels of drug-metabolizing enzymes in control Huh7 cells were generally very low, but DMSO treatment dramatically increased the mRNA levels of most drug-metabolizing enzymes as well as other liver-specific proteins. Importantly, functionality assays confirmed concomitant increases in drug-metabolizing enzyme activity. Additionally, treatment of the Huh7 cells with 3-methylcholanthrene induced cytochrome P450 (CYP) 1A1 expression. The results indicate that DMSO treatment of Huh7 cells profoundly enhances their differentiation state, thus improving the usefulness of this common cell line as an in vitro hepatocyte model.

Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells

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Permissiveness of human hepatoma cell lines for HCV infection

The rate of hepatitis C virus infection initiation in vitro is directly related to particle density

Characterization of increased drug metabolism activity in dimethyl sulfoxide (DMSO)-treated Huh7 hepatoma cells