Arbidol: a broad-spectrum antiviral that inhibits acute and chronic HCV infection

Arbidol (ARB) is an antiviral compound that was originally proven effective for treatment of influenza and several other respiratory viral infections. The broad spectrum of ARB anti-viral activity led us to evaluate its effect on hepatitis C virus (HCV) infection and replication in cell culture. Long-term ARB treatment of Huh7 cells chronically replicating a genomic length genotype 1b replicon resulted in sustained reduction of viral RNA and protein expression, and eventually cured HCV infected cells. Pre-treatment of human hepatoma Huh7.5.1 cells with 15 μM ARB for 24 to 48 hours inhibited acute infection with JFH-1 virus by up to 1000-fold. The inhibitory effect of ARB on HCV was not due to generalized cytotoxicity, nor to augmentation of IFN antiviral signaling pathways, but involved impaired virus-mediated membrane fusion. ARB's affinity for membranes may inhibit several aspects of the HCV lifecycle that are membrane-dependent

Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles via Peptide-Induced Fusion

In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 µm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that ~6 to 10 LUV fused per µm2 of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dyalisis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 µm2/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state

Multiple effects of silymarin on the hepatitis C virus lifecycle

Silymarin, an extract from milk thistle (Silybum marianum), and its purified flavonolignans have been recently shown to inhibit hepatitis C virus (HCV) infection, both in vitro and in vivo. In the current study, we further characterized silymarin's antiviral actions. Silymarin had antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin but not silibinin inhibited genotype 2a NS5B RNA-dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti-HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b BK and four 1b RDRPs derived from HCV-infected patients. Moreover, silymarin did not inhibit HCV replication in five independent genotype 1a, 1b, and 2a replicon cell lines that did not produce infectious virus. Silymarin inhibited microsomal triglyceride transfer protein activity, apolipoprotein B secretion, and infectious virion production into culture supernatants. Silymarin also blocked cell-to-cell spread of virus. CONCLUSION: Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarin's antiviral action appear to include blocking of virus entry and transmission, possibly by targeting the host cell

Mechanism of Inhibition of Enveloped Virus Membrane Fusion by the Antiviral Drug Arbidol

The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection

Differential In Vitro Effects of Intravenous versus Oral Formulations of Silibinin on the HCV Life Cycle and Inflammation

Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV) RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp) with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40–85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43–73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases

Lipoprotein Receptors and Lipid Enzymes in Hepatitis C Virus Entry and Early Steps of Infection

Viruses are obligate intracellular agents that depend on host cells for successful propagation, hijacking cellular machineries to their own profit. The molecular interplay between host factors and invading viruses is a continuous coevolutionary process that determines viral host range and pathogenesis. The hepatitis C virus (HCV) is a strictly human pathogen, causing chronic liver injuries accompanied by lipid disorders. Upon infection, in addition to protein-protein and protein-RNA interactions usual for such a positive-strand RNA virus, HCV relies on protein-lipid interactions at multiple steps of its life cycle to establish persistent infection, making use of hepatic lipid pathways. This paper focuses on lipoproteins in HCV entry and on receptors and enzymes involved in lipid metabolism that HCV exploits to enter hepatocytes

Circulating micro-RNAs as biomarkers of liver fibrosis progression in hepatitis C virus/HIV-1-co-infected patients: a ‘miR’-velous opportunity of early diagnosis?

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Voie de signalisation Hippo, microenvironnement protumoral et carcinome hépato-cellulaire

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Une intégrine participe à la différenciation des cellules souches selon l’élasticité de la matrice extracellulaire

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HCV Virology

International audienceHCV was discovered and its sequence cloned in 1989, the acme of the pioneering work of Michael Houghton's group (Choo et al., 1989). Since then, studies of the HCV replication cycle have long been hampered by the following facts : (i) HCV is a strictly human pathogen that causes a chronic infection; (ii) the target cells of HCV replication are solely hepatocytes; (iii) virions isolated from the serum of HCVpositive patients could not infect cell culture systems in a productive manner; (iv) viral titration (assay of viral proteins and/or genome) could not be related to infectivity. Chimpanzees are permissive to HCV, but their use comes at prohibitive costs, and features of the disease are not similar to human clinical signs. Three major breakthroughs allowed to overcome these restrictions : the first one came with the discovery and implementation of the HCV replicon system in cell culture based on a full-length consensus genome from a viral RNA isolated from an infected human liver. This genome was then used to construct subgenomic replicons, which, upon transfection into a human hepatoma cell line, replicated to high levels (Lohmann et al., 1999). With this invaluable tool, scientists in the field of hepatitis C were able to address fundamental questions related to viral replication in vitro, and this also fuelled intensive research on antiviral molecules. The second breakthrough came with the molecular design of viral particles able to mimic the first stages of the interaction of HCV with its target cells: virion attachment to the cell surface, recognition of specific receptor molecules, internalization in intracellular compartments, viral fusion. This tool, so-called HCV pseudoparticles (HCVpp), was based on retroviral capsids harboring the glycoproteins E1 and E2 of HCV at the virion surface. HCVpp were subsequently used to study viral entry, fusion and humoral immune responses and to dissect the molecular details of the steps by which HCV initiated infection (Bartosch et al., 2003a). The third breakthrough emerged from a clinical case report in Japan of a fulminant hepatitis caused by HCV. This unusual feature for HCV led to the isolation and discovery of a new HCV clone, displaying a full replication cycle in cell culture settings. This clone was called JFH-1 (for Japanese Fulminant Hepatitis), and produced infectious viral particles capable of several rounds of in vitro infection, called HCVcc (HCV grown in Cell Culture) (Wakita et al., 2005, Lindenbach et al., 2005). These particles are used for structural and functional studies, and were successfully applied to the discovery of antiviral therapies now licensed and capable of HCV eradication