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Hepatitis C virus (HCV) remains a serious global health problem that lacks an effective cure. Although the introduction of protease inhibitors to the current standard-of-care interferon/ribavirin therapy for HCV infection has improved sustained virological response of genotype 1-infected patients, these inhibitors exacerbate already problematic side effects. Thus, new HCV antivirals are urgently needed. Using a cell-protection screen previously developed in our laboratory, we evaluated 30,426 compounds for inhibitors of potentially any stage of the HCV life cycle and identified 49 new HCV inhibitors. The two most potent hits, hydroxyzine and chlorcyclizine, belong to the family of benzhydrylpiperazines and were found to inhibit the entry of cell culture-produced HCV with IC50 values of 19 and 2.3 nM, respectively, and therapeutic indices of >500 and >6500. Both compounds block HCV entry at a late stage of entry prior to viral fusion and their inhibitory activities are highly dependent on the host's cholesterol content. Both compounds are currently used in the clinic for treating allergy-related disorders and the reported peak plasma level (160 nM) and estimated liver concentration (1.7 μM) of hydroxyzine in humans are much higher than the molecule's anti-HCV IC90 in cell culture (64 nM). Further studies are therefore justified to evaluate the use of these molecules in an anti-HCV therapeutic regimen. [ABSTRACT FROM AUTHOR]

Abstract: Viruses have evolved to complex relationship with their host cells. Many viruses modulate the lipid composition, lipid synthesis and signaling of their host cell. Lipids are also an essential part of the life cycle of the hepatitis C virus (HCV). HCV is a major human pathogen, persistently infecting 170 million people worldwide, with no currently effective treatment available for all patients. HCV appears to make use of the host lipid metabolism and one common feature of chronic hepatitis C is the steatosis, characterized by excessive accumulation of triglycerides and lipid content in the liver. Thus, HCV lifecycle appears to be closely connected to host cell lipid metabolism, from cell entry, through viral RNA replication to viral particle production and formation/assembly. HCV particles have a unique lipid composition, certainly distinct from other viruses. In the blood of chronically-infected patients, viral particles are bound to serum lipoproteins and are thus called lipo-viro-particles. The density of these circulating viral particles is heterogeneous. Specific infectivity and fusion of low density particles are greater than those of high density particles. Lipids and association to lipoproteins therefore play a key role in HCV life cycle. The purpose of this review is to make a state of the art on recent findings on the contribution of lipids in cell entry and membrane fusion of HCV. The influence of lipids as chemically-defined entities will be analyzed, as well as the role played by cholesterol transporters and lipoprotein receptors in HCV entry and fusion. Since viral entry would constitute a key target for antiviral strategies, inhibitor molecules interacting with viral and/or cellular membranes or interfering with the function of lipid metabolism regulators of HCV entry could offer strong antiviral potential. This will be lastly discussed in this review. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The density of hepatitis C virus (HCV) particles circulating in the blood of chronically infected patients and of cell-culture produced HCV is heterogeneous. Specific infectivity and fusion of low density particles are higher than those of high density particles. We recently characterized hybrid particles produced by Caco-2 colon or Huh-7.5 liver cells transduced with HCV E1 and E2 envelope glycoproteins. Caco-2-derived particles, called empty lipo-viral particles (eLVP), are composed of triglyceride-rich lipoproteins positive for apolipoproteins B (i.e. apoB100 and apoB48) and contain HCV E1 and E2. Here we aimed at characterizing the morphology and in vitro fusion properties of eLVP using electron microscopy and fluorescence spectroscopy. They displayed the aspect of β-lipoproteins, and immunogold labeling confirmed the presence of apoB and HCV E1 and E2 at their surface. These particles are able to fuse with lipid bilayers (liposomes) in a fusion process leading to the coalescence of internal contents of triglyceride-rich lipoproteins particles and liposomes. Fusion was pH-dependent and could be inhibited by either Z-fFG, a peptide known to inhibit viral fusion, or by monoclonal antibodies directed against HCV E2 or the apolipoprotein moiety of the hybrid particle. Interestingly, particles derived from Huh-7.5 cells failed to display equivalent efficient fusion. Optimal fusion activity is, thus, observed when HCV envelope proteins are associated to apoB-positive hybrid particles. Our results, therefore, point to a crucial role of the E1 and E2 proteins in HCV fusion with a subtle interplay with the apolipoprotein part of eLVP. [ABSTRACT FROM AUTHOR]

Enveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called “rafts”, or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class. [ABSTRACT FROM AUTHOR]

Hepatitis C affects ∼3% of the world population, yet its current treatment options are limited to interferon-ribavirin drug regimens which achieve a 50-70% cure rate depending on the hepatitis C virus (HCV) genotype. Besides extensive screening for HCV-specific compounds, some well-established medicinal drugs have recently demonstrated an anti-HCV effect in HCV replicon cells. One of these drugs is arbidol (ARB), a Russian-made broad-spectrum antiviral agent, which we have previously shown to inhibit acute and chronic HCV infection. Here we show that ARB inhibits the cell entry of HCV pseudoparticles of genotypes 1a, 1b, and 2a in a dose-dependent fashion. ARB also displayed a dose-dependent inhibition of HCV membrane fusion, as assayed by using HCV pseudoparticles (HCVpp) and fluorescent liposomes. ARB inhibition of HCVpp fusion was found to be more effective on genotype 1a than on genotypes 1b and 2a. In vitro biochemical studies revealed association of ARB with membranelike environments such as detergents and with lipid membranes. This association was particularly prominent at acidic pH which is optimal for HCV-mediated fusion. Our results suggest that the affinity of ARB for lipid membranes could account for its anti-HCV actions, together with a differential level of interaction with key motifs in HCV glycoproteins of different genotypes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

PtdIns(3,5)P2 is required for cargo-selective sorting to the vacuolar lumen via the multivesicular body (MVB). Here we show that Ent3p, a yeast epsin N-terminal homology (ENTH) domain-containing protein, is a specific PtdIns(3,5)P2 effector localized to endosomes. The ENTH domain of Ent3p is essential for its PtdIns(3,5)P2 binding activity and for its membrane interaction in vitro and in vivo. Ent3p is required for protein sorting into the MVB but not for the internalization step of endocytosis. Ent3p is associated with clathrin and is necessary for normal actin cytoskeleton organization. Our results show that Ent3p is required for protein sorting into intralumenal vesicles of the MVB through PtdIns(3,5)P2 binding via its ENTH domain. [Copyright &y& Elsevier]

Examines the role of phospholipid species on mediated fusion of golgi membranes. Causes of dramatic conformation rearrangements of membranes; Exposure of the buried hydrophobic sites; Reassembly of Golgi cisternae after pharmacological or physiological vesiculation.

The article discusses a study related to correlation between curcumin hepatitis C virus infection. It states that hepatitis C is a health problem with maor inequalities in the access to healthcare and implementation of treatments between regions. It further informs that HCV is an enveloped RNA virus classified into seven genotypes, and virions are found associated to beta-lipoproteins in the serum of patients.

Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes. Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae. Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH. [ABSTRACT FROM AUTHOR]

Over time, chronic HCV infection can lead to hepatocellular carcinoma (HCC), a process that involves changes to the liver extracellular matrix (ECM). However, the exact mechanisms by which HCV induces HCC remain unclear. Therefore, we sought to investigate the impact of HCV on the liver ECM, with a focus on heparanase-1 (HPSE). HPSE expression was assessed by quantitative reverse-transcription PCR, immunoblotting and immunofluorescence in liver biopsies infected or not with HCV, and in 10-day-infected hepatoma Huh7.5 cells. Cell lines deficient for or overexpressing HPSE were established to study its role during infection. HCV propagation led to significant HPSE induction, in vivo and in vitro. HPSE enhanced infection when exogenously expressed or supplemented as a recombinant protein. Conversely, when HPSE expression was downregulated or its activity blocked, HCV infection dropped, suggesting a role of HPSE in the HCV life cycle. We further studied the underlying mechanisms of such observations and found that HPSE favored HCV release by enhancing CD63 synthesis and exosome secretion, but not by stimulating HCV entry or genome replication. We also showed that virus-induced oxidative stress was involved in HPSE induction, most likely through NF-κB activation. We report for the first time that HCV infection is favored by HPSE, and upregulates HPSE expression and secretion, which may result in pathogenic alterations of the ECM. Chronic hepatitis C virus (HCV) infection can lead to hepatocellular carcinoma development in a process that involves derangement of the extracellular matrix (ECM). Herein, we show that heparanase-1, a protein involved in ECM degradation and remodeling, favors HCV infection and is upregulated by HCV infection; this upregulation may result in pathogenic alterations of the ECM. [Display omitted] • Heparanase-1 (HPSE) is upregulated by HCV in an NF-κB-dependent manner. • HPSE favors HCV replication cycle at the virus release step. • HCV release is dependent on the tetraspanin CD63. • High HPSE levels may favor pathologic liver alterations. [ABSTRACT FROM AUTHOR]

Highlights: [•] Arbidol (ARB) is licensed in Russia and China for the treatment of influenza and other viral infections. [•] ARB inhibits a large panel of viral pathogens, enveloped or not. [•] ARB displays a dual binding activity to lipid membranes and to viral or cellular proteins. [•] It blocks viral endocytosis and replication in membranous intracellular compartments. [Copyright &y& Elsevier]

In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-ß-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produce VLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs. [ABSTRACT FROM AUTHOR]

The entry of enveloped viruses into their host cells involves several successive steps, each one being amenable to therapeutic intervention. Entry inhibitors act by targeting viral and/or cellular components, through either the inhibition of protein-protein interactions within the viral envelope proteins or between viral proteins and host cell receptors, or through the inhibition of protein-lipid interactions. Interestingly, inhibitors that concentrate into/onto the membrane in order to target a protein involved in the entry process, such as arbidol or peptide inhibitors of the human immunodeficiency virus (HIV), could allow the use of doses compatible with therapeutic requirements. The efficacy of these drugs validates entry as a point of intervention in viral life cycles. Strategies based upon small molecule antiviral agents, peptides, proteins or nucleic acids, would most likely prove efficient in multidrug combinations, in order to inhibit several steps of virus life cycle and prevent disease progression. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus of about 9.6 kb. Like all enveloped viruses, the HCV membrane fuses with the host cell membrane during the entry process and thereby releases the genome into the cytoplasm, initiating the viral replication cycle. To investigate the features of HCV membrane fusion, we developed an in vitro fusion assay using cell culture-produced HCV and fluorescently labeled liposomes. With this model we could show that HCV-mediated fusion can be triggered in a receptor-independent but pH-dependent manner and that fusion of the HCV particles with liposomes is dependent on the viral dose and on the lipid composition of the target membranes. In addition CBH-5, an HCV E2-specific antibody, inhibited fusion in a dose-dependent manner. Interestingly, point mutations in E2, known to abrogate HCV glycoprotein-mediated fusion in a cell-based assay, altered or even abolished fusion in the liposome-based assay. When assaying the fusion properties of HCV particles with different buoyant density, we noted higher fusogenicity of particles with lower density. This could be attributable to inherently different properties of low density particles, to association of these particles with factors stimulating fusion, or to co-floatation of factors enhancing fusion activity in trans. Taken together, these data show the important role of lipids of both the viral and target membranes in HCV-mediated fusion, point to a crucial role played by the E2 glycoprotein in the process of HCV fusion, and reveal an important behavior of HCV of different densities with regard to fusion. [ABSTRACT FROM AUTHOR]

Gold nanoparticles (AuNPs) have demonstrated outstanding performance in many biomedical applications. Their safety is recognised; however, their effects on the immune system remain ill defined. Antigen-presenting cells (APCs) are immune cells specialised in sensing external stimulus and in capturing exogenous materials then delivering signals for the immune responses. We used primary macrophages (Ms) and dendritic cells (DCs) of mice as an APC model. Whereas AuNPs did not alter significantly Ms and DCs functions, the exposure to AuNPs affected differently Ms and DCs in their responses to subsequent stimulations. The secretion of inflammatory molecules like cytokines (IL-6, TNF-α), chemokine (MCP-1), and reactive oxygen species (ROS) were altered differently in Ms and DCs. Furthermore, the metabolic activity of Ms was affected with the increase of mitochondrial respiration and glycolysis, while only a minor effect was seen on DCs. Antigen presentation to T cells increased when DCs were exposed to AuNPs leading to stronger Th1, Th2, and Th17 responses. In conclusion, our data provide new insights into the complexity of the effects of AuNPs on the immune system. Although AuNPs may be considered as devoid of significant effect, they may induce discrete modifications on some functions that can differ among the immune cells. [ABSTRACT FROM AUTHOR]

Abstract Fibrosis is a serious health problem often leading to accompanying organ failure. During the manifestation of the disease, an accumulation of different extracellular matrix (ECM) molecules, such as proteoglycans, takes place. There is no appropriate therapeutic option available to heal fibrosis to date. Current research focuses primarily on targets such as the cytokine transforming growth factor-β1 (TGF-β1), which is assumed to be one of the key mediators of fibrosis. Both xylosyltransferase isoforms, XT-I and XT-II, catalyze the rate-limiting step of the proteoglycan biosynthesis. Consequently, inhibiting XT activity could be a promising approach to treat fibrosis. It was shown in earlier studies that nucleotides and nucleosides have anti-fibrotic properties and decrease XT activity in cell-free systems. In contrast, we evaluated the mechanisms beyond an UDP-mediated induction of intracellular XT-activity in normal human dermal fibroblasts (NHDF). The observed pseudo-fibrotic XT increasement could be attributed to a compensation of decreased UDP-glucuronate decarboxylase 1 (UXS1) mRNA expression as well as a diminished intracellular UDP-xylose concentration. In summary, our results describe a so far unknown XT-inductive pathway and show that UDP could be a promising molecule for the development of an anti-fibrotic therapy. Nevertheless, XT activity has to be inhibited in parallel intracellularly. Graphical abstract Image 1 Highlights • UDP treatment of NHDF leads to increased intracellular XT-activity. • UDP treatment results in decreased gene expression levels of ECM-associated targets. • UXS1 knockdown results in increased XT activity and XYLT1 expression. • Anti-fibrotic UDP treatment must be complemented by XT inhibition. [ABSTRACT FROM AUTHOR]

The hepatitis C virus (HCV) infects hepatocytes after binding to heparan sulfate proteoglycans, in particular Syndecan-1, followed by recognition of the tetraspanin CD81 and other receptors. Heparan sulfate proteoglycans are found in a specific microenvironment coating the hepatocyte surface called the glycocalyx and are receptors for extracellular matrix proteins, cytokines, growth factors, lipoproteins, and infectious agents. We investigated the mutual influence of HCV infection on the glycocalyx and revealed new links between Syndecan-1 and CD81. Hepatocyte infection by HCV was inhibited after knocking down Syndecan-1 or Xylosyltransferase 2, a key enzyme of Syndecan-1 biosynthesis. Simultaneous knockdown of Syndecan-1 and CD81 strongly inhibited infection, suggesting their cooperative action. At early infection stages, Syndecan-1 and virions colocalized at the plasma membrane and were internalized in endosomes. Direct interactions between Syndecan-1 and CD81 were revealed in primary and transformed hepatocytes by immunoprecipitation and proximity ligation assays. Expression of Syndecan-1 and Xylosyltransferase 2 was altered within days post-infection, and the remaining Syndecan-1 pool colocalized poorly with CD81. The data indicate a profound reshuffling of the hepatocyte glycocalyx during HCV infection, possibly required for establishing optimal conditions of viral propagation. [ABSTRACT FROM AUTHOR]

Objective: Inflammation and oxidative stress drive disease progression in chronic hepatitis C (CHC) towards hepatocellular carcinoma. HCV is known to increase intracellular levels of reactive oxygen species (ROS), but how it eliminates ROS is less well known. The role of the ROS scavenger glutathione peroxidase 4 (GPx4), induced by HCV, in the viral life cycle was analysed. Design: The study was performed using a replicative in vitro HCV infection model and liver biopsies derived from two different CHC patient cohorts. Results: A screen for HCV-induced peroxide scavengers identified GPx4 as a host factor required for HCV infection. The physiological role of GPx4 is the elimination of lipid peroxides from membranes or lipoproteins. GPx4-silencing reduced the specific infectivity of HCV by up to 10-fold. Loss of infectivity correlated with 70% reduced fusogenic activity of virions in liposome fusion assays. NS5A was identified as the protein that mediates GPx4 induction in a phosphatidylinositol-3-kinase-dependent manner. Levels of GPx4 mRNA were found increased in vitro and in CHC compared with control liver biopsies. Upon successful viral eradication, GPx4 transcript levels returned to baseline in vitro and also in the liver of patients. Conclusions: HCV induces oxidative stress but controls it tightly by inducing ROS scavengers. Among these, GPx4 plays an essential role in the HCV life cycle. Modulating oxidative stress in CHC by specifically targeting GPx4 may lower specific infectivity of virions and prevent hepatocarcinogenesis, especially in patients who remain difficult to be treated in the new era of interferon-free regimens. [ABSTRACT FROM AUTHOR]

In hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1-TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein. [ABSTRACT FROM AUTHOR]

Objective Adult primary human hepatocytes (PHHs) support the complete infection cycle of natural HCV from patients' sera. The molecular details underlying sera infectivity towards these cells remain largely unknown. Therefore, we sought to gain a deeper comprehension of these features in the most physiologically relevant culture system. Design Using kinetic experiments, we defined the optimal conditions to infect PHH and explored the link between cell organisation and permissivity. Based on their infectivity, about 120 sera were classified in three groups. Concentration of 52 analytes was measured in 79 selected sera using multiplexed immunobead-based analyte profiling. Results PHH permissivity towards HCV infection negatively correlated with cell polarisation and formation of functional bile canaliculi. PHH supported HCV replication for at least 2 weeks with de novo virus production. Depending on their reactivity, sera could be classified in three groups of high, intermediate or low infectivity toward PHH. Infectivity could not be predicted based on the donors' clinical characteristics, viral load or genotype. Interestingly, highly infectious sera displayed a specific cytokine profile with low levels of most of the 52 tested analytes. Among them, 24 cytokines/growth factors could impact hepatocyte biology and infection efficiency. Conclusions We identified critical factors leading to efficient PHH infection by HCV sera in vitro. Overall, we showed that this cellular model provides a useful tool for studying the mechanism of HCV infection in its natural host cell, selecting highly infectious isolates, and determining the potency of drugs towards various HCV strains. [ABSTRACT FROM AUTHOR]

Hepatitis C virus ( HCV) is a global health concern infecting 170 million people worldwide. Previous studies indicate that the extract from milk thistle known as silymarin and its main component silibinin inhibit HCV infection. Here we investigated the mechanism of anti- HCV action ofsilymarin-derived compounds at the molecular level. By using live-cell confocal imaging, single particle tracking, transmission electron microscopy and biochemical approaches on HCV-infected human hepatoma cells and primary hepatocytes, we show that silibinin potently inhibits HCV infection and hinders HCV entry by slowing down trafficking through clathrin-coated pits and vesicles. Detailed analyses revealed that silibinin altered the formation of both clathrin-coated pits and vesicles in cells and caused abnormal uptake and trafficking of transferrin, a well-known cargo of the clathrin endocytic pathway. Silibinin also inhibited infection by other viruses that enter cells by clathrin-mediated endocytosis including reovirus, vesicular stomatitis and influenza viruses. Our study demonstrates that silibinin inhibits HCV early steps of infection by affecting endosomal trafficking of virions. It provides new insights into the molecular mechanisms of action of silibinin against HCV entry and also suggests that silibinin is a potential broad-spectrum antiviral therapy. [ABSTRACT FROM AUTHOR]

Highlights: [•] Arbidol is a broad-spectrum antiviral known as an entry and fusion inhibitor. [•] We investigate its mechanism of action on cells by live-cell confocal imaging. [•] Arbidol affects clathrin-mediated endocytosis. [•] This leads to confinement of viral particles in clathrin-coated vesicles. [•] Arbidol acts by impeding dynamin-induced membrane scission. [Copyright &y& Elsevier]

Abstract: Hydrophobic photoaffinity labeling is a powerful strategy to identify hydrophobic segments within molecules, in particular membrane proteins. Here we report the design and synthesis of a novel family of fluorescent and photosensitive lipid tools, which have a common amino acid scaffold functionalized by three groups: (i) a first fatty acid chain grafted with a photoactivatable benzophenone moiety (Fatty Acid BenzoPhenone, FABP), (ii) a second fatty acid chain to ensure anchoring into a half-bilayer or hydrophobic environment, and (iii) a fluorescent carboxytetramethylrhodamine headgroup (CTMR) to detect the photolabeled compound. We present data of the synthesis and characterization of three lipid tools whose benzophenone ring is situated at various distances from the central scaffold. We could therefore establish structure/properties relationships dependent upon the depth of insertion of benzophenone into the membrane. Our lipid tools were extensively characterized both physico- and bio-chemically, and we assessed their functionality in vitro using bacterioRhodopsin (bR). We thus provide the scientific community with novel and reliable tools for the identification and study of hydrophobic regions in proteins. [Copyright &y& Elsevier]

A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches. [ABSTRACT FROM AUTHOR]

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 μ. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The cell entry and humoral immune response of the human pathogen Lassa virus (LV), a biosafety level 4 (BSL4) Old World arenavirus, are not well characterized. LV pseudoparticles (LVpp) are a surrogate model system that has been used to decipher factors and routes involved in LV cell entry under BSL2 conditions. Here, we describe LVpp, which are highly infectious, with titers approaching those obtained with pseudoparticles displaying G protein of vesicular stomatitis virus and their the use for the characterization of LV cell entry and neutralization. Upon cell attachment, LVpp utilize endocytic vesicles for cell entry as described for many pH-dependent viruses. However, the fusion of the LV glycoproteins is activated at unusually low pH values, with optimal fusion occurring between pH 4.5 and 3, a pH range at which fusion characteristics of viral glycoproteins have so far remained largely unexplored. Consistent with a shifted pH optimum for fusion activation, we found wild-type LV and LVpp to display a remarkable resistance to exposure to low pH. Finally, LVpp allow the fast and quantifiable detection of neutralizing antibodies in human and animal sera and will thus facilitate the study of the humoral immune response in LV infections. [ABSTRACT FROM AUTHOR]

Cell entry of hepatitis C virus (HCV) is strikingly linked to lipoproteins and their receptors. Particularly, high density lipoprotein (HDL) enhances infectivity of HCV by involving the lipid-transfer function of the scavenger receptor BI, a receptor for both HDL and HCV. Here, we demonstrate that apoC-I, an exchangeable apolipoprotein that predominantly resides in HDL, specifically enhances HCVcc and HCVpp infectivity and increases the fusion rates between viral and target membranes via a direct interaction with the HCV surface. We identify the hypervariable region 1, located at the N terminus of the HCV E2 glycoprotein, as an essential viral component that modulates apoC-I-mediated enhancement of HCV fusion properties. The affinity of apoC-I for the HCV membrane may predispose it for fusion with a target membrane via alterations of its outer phospholipid layer. Conversely, we found that excess apoC-I provided as lipoprotein-free protein induces the disruption of the HCV membrane and subsequent loss of infectivity. Furthermore, our data indicate that HDL neither interacts nor spontaneously exchanges apoC-I with HCV virions. Because apoC-I is not present in serum as a lipoprotein-free form, our results suggest that HDL-embedded apoC-I could be released from the lipoprotein particle through a fine-tuned mechanism regulated via a triple interplay between hypervariable region 1, HDL, and scavenger receptor BI, resulting in optimal apoC-I recruitment on the viral membrane. These results provide the first description of a host serum factor helping the fusion process of an enveloped virus. [ABSTRACT FROM AUTHOR]

It has been suggested that the hepatitis C virus (HCV) infects host cells through a pH-dependent internalization mechanism, but the steps leading from virus attachment to the fusion of viral and cellular membranes remain uncharacterized. Here we studied the mechanism underlying the HCV fusion process in vitro using liposomes and our recently described HCV pseudoparticles (pp) bearing functional E1E2 envelope glycoproteins. The fusion of HCVpp with liposomes was monitored with fluorescent probes incorporated into either the HCVpp or the liposomes. To validate these assays, pseudoparticles bearing either the hemagglutinin of the influenza virus or the amphotropic glycoprotein of murine leukemia virus were used as models for pH-dependent and pH-independent entry, respectively. The use of assays based either on fusion-induced dequenching of fluorescent probes or on reporter systems, which produce fluorescence when the virus and liposome contents are mixed, allowed us to demonstrate that HCVpp mediated a complete fusion process, leading to the merging of both membrane leaflets and to the mixing of the internal contents of pseudoparticle and liposome. This HCVpp-mediated fusion was dependent on low pH, with a threshold of 6.3 and an optimum at about 5.5. Fusion was temperature-dependent and did not require any protein or receptor at the surface of the target liposomes. Most interestingly, fusion was facilitated by the presence of cholesterol in the target membrane. These findings clearly indicate that HCV infection is mediated by a pH-dependent membrane fusion process. This paves the way for future studies of the mechanisms underlying HCV membrane fusion. [ABSTRACT FROM AUTHOR]

Simple Summary: In the era of direct-acting antivirals against the hepatitis C virus (HCV), curing chronic hepatitis C has become a reality. However, while replicating chronically, HCV creates a peculiar state of inflammation and oxidative stress in the infected liver, which fuels DNA damage at the onset of HCV-induced hepatocellular carcinoma (HCC). This cancer, the second leading cause of death by cancer, remains of bad prognosis when diagnosed. This review aims to decipher how HCV durably alters elements of the extracellular matrix that compose the liver microenvironment, directly through its viral proteins or indirectly through the induction of cytokine secretion, thereby leading to liver fibrosis, cirrhosis, and, ultimately, HCC. Chronic infection by the hepatitis C virus (HCV) is a major cause of liver diseases, predisposing to fibrosis and hepatocellular carcinoma. Liver fibrosis is characterized by an overly abundant accumulation of components of the hepatic extracellular matrix, such as collagen and elastin, with consequences on the properties of this microenvironment and cancer initiation and growth. This review will provide an update on mechanistic concepts of HCV-related liver fibrosis/cirrhosis and early stages of carcinogenesis, with a dissection of the molecular details of the crosstalk during disease progression between hepatocytes, the extracellular matrix, and hepatic stellate cells. [ABSTRACT FROM AUTHOR]

Direct stochastic optical reconstruction microscopy (dSTORM), developed in the last decade, has revolutionised optical microscopy by enabling scientists to visualise objects beyond the resolution provided by conventional microscopy (200 nm). We developed an innovative method based on blinking particle standards and conditions for long-lived imaging over several weeks. Stable localisation precisions within the 10 nm-range were achieved for single virions and in cellulo 2D imaging of centrosomes, as well as their reliable reconstruction in 3D dSTORM. [ABSTRACT FROM AUTHOR]

The variability of the hepatitis C virus (HCV), which likely contributes to immune escape, is most pronounced in hypervariable region 1 (HVR1) of viral envelope protein 2. This domain is the target for neutralizing antibodies, and its deletion attenuates replication in vivo. Here we characterized the relevance of HVR1 for virus replication in vitro using cell culture-derived HCV. We show that HVR1 is dispensable for RNA replication. However, viruses lacking HVR1 (ΔHVR1) are less infectious, and separation by density gradients revealed that the population of HVR1 virions comprises fewer particles with low density. Strikingly, ΔHVR1 particles with intermediate density (1.12 g/ml) are as infectious as wild-type virions, while those with low density (1.02 to 1.08 g/ml) are poorly infectious, despite quantities of RNA and core similar to those in wild-type particles. Moreover, ΔHVR1 particles exhibited impaired fusion, a defect that was partially restored by an E1 mutation (I347L), which also rescues infectivity and which was selected during long-term culture. Finally, ΔHVR1 particles were no longer neutralized by SR-B1-specific immunoglobulins but were more prone to neutralization and precipitation by soluble CD81, E2-specific monoclonal antibodies, and patient sera. These results suggest that HVR1 influences the biophysical properties of released viruses and that this domain is particularly important for infectivity of low-density particles. Moreover, they indicate that HVR1 obstructs the viral CD81 binding site and conserved neutralizing epitopes. These functions likely optimize virus replication, facilitate immune escape, and thus foster establishment and maintenance of a chronic infection. [ABSTRACT FROM AUTHOR]

The transmembrane (TM) domains of hepatitis C virus (HCV) envelope glycoproteins E1 and E2 have been shown to play multiple roles during the biogenesis of the E1E2 heterodimer. By using alanine scanning insertion mutagenesis within the TM domains of HCV envelope glycoproteins, we have previously shown that the central regions of these domains as well as the N-terminal part of the TM domain of E1 are involved in heterodimerization. Here, we used a tryptophan replacement scan of these regions to identify individual residues that participate in those interactions. Our mutagenesis study identified at least four residues involved in heterodimerization: Gly 354, Gly 358, Lys 370, and Asp 728. Interestingly, Gly 354 and Gly 358 belong to a GXXXG oligomerization motif. Our tryptophan mutants were also used to generate retrovirus-based, HCV-pseudotyped particles (HCVpp) in order to analyze the effects of these mutations on virus entry. Surprisingly, two mutants consistently displayed higher infectivity compared to that of the wild type. In contrast, HCVpp infectivity was strongly affected for many mutants, despite normal E1E2 heterodimerization and normal levels of incorporation of HCV glycoproteins into HCVpp. The characterization of some of these HCVpp mutants in the recently developed in vitro fusion assay using fluorescent-labeled liposomes indicated that mutations reducing HCVpp infectivity without altering E1E2 heterodimerization affected the fusion properties of HCV envelope glycoproteins. In conclusion, this mutational analysis identified residues involved in E1E2 heterodimerization and revealed that the TM domains of HCV envelope glycoproteins play a major role in the fusion properties of these proteins. [ABSTRACT FROM AUTHOR]