Hepatitis C virus cell entry : role of lipoproteins and cellular receptors

Hepatitis C virus (HCV), a major cause of chronic liver disease, is a single-stranded positive sense virus of the family Flaviviridae. HCV cell entry is a multi-step process, involving several viral and cellular factors that trigger virus uptake into the hepatocyte. Tetraspanin CD81, human scavenger receptor SR-BI, and tight junction molecules Claudin-1 and occludin are the main receptors that mediate HCV entry. In addition, the virus may use glycosaminoglycans and/or low density receptors on host cells as initial attachment factors. A unique feature of HCV is the dependence of virus replication and assembly on host cell lipid metabolism. Most notably, during HCV assembly and release from the infected cells, virus particles associate with lipids and very-low-density lipoproteins. Thus, infectious virus circulates in patient sera in the form of triglyceride-rich particles. Consequently, lipoproteins and lipoprotein receptors play an essential role in virus uptake and the initiation of infection. This review summarizes the current knowledge about HCV receptors, mechanisms of HCV cell entry and the role of lipoproteins in this process

Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified

Up-regulation of the ATP-binding cassette transporter A1 inhibits hepatitis C virus infection.

International audienceHepatitis C virus (HCV) establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL) particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR) agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts). The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis, leading to the inhibition of virus-cell fusion and thus HCV cell entry. Therefore besides other beneficial roles, ABCA1 might represent a potential target for HCV therapy

Hepatitis C Virus Controls Interferon Production through PKR Activation

Hepatitis C virus is a poor inducer of interferon (IFN), although its structured viral RNA can bind the RNA helicase RIG-I, and activate the IFN-induction pathway. Low IFN induction has been attributed to HCV NS3/4A protease-mediated cleavage of the mitochondria-adapter MAVS. Here, we have investigated the early events of IFN induction upon HCV infection, using the cell-cultured HCV JFH1 strain and the new HCV-permissive hepatoma-derived Huh7.25.CD81 cell subclone. These cells depend on ectopic expression of the RIG-I ubiquitinating enzyme TRIM25 to induce IFN through the RIG-I/MAVS pathway. We observed induction of IFN during the first 12 hrs of HCV infection, after which a decline occurred which was more abrupt at the protein than at the RNA level, revealing a novel HCV-mediated control of IFN induction at the level of translation. The cellular protein kinase PKR is an important regulator of translation, through the phosphorylation of its substrate the eIF2α initiation factor. A comparison of the expression of luciferase placed under the control of an eIF2α-dependent (IRESEMCV) or independent (IRESHCV) RNA showed a specific HCV-mediated inhibition of eIF2α-dependent translation. We demonstrated that HCV infection triggers the phosphorylation of both PKR and eIF2α at 12 and 15 hrs post-infection. PKR silencing, as well as treatment with PKR pharmacological inhibitors, restored IFN induction in JFH1-infected cells, at least until 18 hrs post-infection, at which time a decrease in IFN expression could be attributed to NS3/4A-mediated MAVS cleavage. Importantly, both PKR silencing and PKR inhibitors led to inhibition of HCV yields in cells that express functional RIG-I/MAVS. In conclusion, here we provide the first evidence that HCV uses PKR to restrain its ability to induce IFN through the RIG-I/MAVS pathway. This opens up new possibilities to assay PKR chemical inhibitors for their potential to boost innate immunity in HCV infection

Rôle des lipoprotéines associées au virus de l'hépatite C et des microtubules dans l'entrée du virus dans la cellule et l'établissement de l'infection

Le VHC reste un problème majeur de santé publique. Malgré la mise au point d un modèle de réplication in vitro, les mécanismes conduisant à l infection restent encore méconnus. Le VHC est sécrété et circule dans le sérum associé à des lipoprotéines. L importance de celles-ci pour le cycle viral nous a conduits à étudier le rôle de la LPL, une enzyme lipolytique, dans l infection par le VHC. La LPL potentialise l attachement et l internalisation du VHC par un mécanisme similaire à la clearance hépatique des lipoprotéines. La LPL dimérique forme un pont entre les lipoprotéines associées au VHC et les HSPG à la surface des cellules. Son action conduit à une inhibition de l infection par les souches virales JFH-1 et J6/JFH-1 produites en culture cellulaire et chez les souris uPA-SCID. L analyse par ultracentrifugation en gradient d iodixanol des virus produits in vitro et in vivo a montré la présence de 2 populations virales : la première, de densité très faible, est beaucoup plus infectieuse que la seconde, de densité plus élevée. L infection in vitro par ces 2 populations est inhibée par la LPL. Nous avons démontré qu un réseau de microtubules intact et dynamique est crucial pour l entrée du VHC et les étapes menant à l infection. Nous avons mis en évidence une interaction de la protéine de capside avec la tubuline conduisant à une augmentation de la polymérisation des microtubules. Cela suggère que le VHC pourrait utiliser les mécanismes de polymérisation des microtubules pour établir l infection et la capside jouer un rôle essentiel dans ce processus. Les nouvelles approches antivirales pourraient cibler les éléments du cytosquelette et/ou des lipoprotéines associées au VHC.Hepatitis C virus (HCV) infection remains a major health problem. Despite of the development of a cell culture system permitting HCV replication in vitro, the mechanisms leading to infection are still not well understood. HCV is secreted and circulates in the patient serum in association with lipoproteins. The importance of lipoproteins for the virus life cycle led us to investigate a role of lipoprotein lipase (LPL), a lipolytic enzyme, in the cell infection with HCV. We showed that LPL increases virus attachment and internalisation by mechanisms similar to the hepatic uptake of lipoproteins. Dimeric LPL forms a bridge between virus-associated lipoproteins and HSPG at the cell surface. Nevertheless, LPL inhibits cell infection with two virus strains: JFH-1 and J6/JFH-1, produced either in cell culture, or in chimeric uPA-SCID mice with transplanted human hepatocytes. Analyses of the viruses produced in vitro and in vivo in iodixanol gradients showed the presence of two virus populations banding at very low and higher density, the first population being much more infectious than the latter one. Cell infection with low and higher density viruses was inhibited by LPL. Thus, LPL represents a novel inhibitor of HCV infectivity.We showed that the intact and dynamic microtubules are crucial for HCV cell entry and early post-fusion steps, leading to productive infection. In addition, we provided evidence that HCV core protein directly interacts with a/b tubulin, increasing polymerisation of microtubules. These observations suggest that HCV could use microtubule polymerisation mechanisms to establish infection, in which virus core protein might play an important role.New anti-viral approaches may thus target the elements of the cytoskeleton and/or lipoproteins associated with virus particles.PARIS-BIUP (751062107) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Evaluation of core and NS4B synthetic peptide-based immunoassays for the detection of hepatitis C virus antibodies in clinical samples from Cameroon, Central Africa.

International audienceBACKGROUND: According to previous data, the antibodies produced during natural hepatitis C virus (HCV) infection frequently recognize amino acids 10-43 in the core protein and 1689-1740 or 1921-1940 in the non-structural 4B (NS4B) protein. The reactivity of these peptides with the corresponding antibodies has mainly been evaluated using serum samples from Western countries where HCV genotype 1 (HCV-1) is predominant, and no information is available concerning samples from sub-Saharan countries where high HCV variability has been reported. OBJECTIVE OF THIS STUDY: To evaluate the performance of HCV core and NS4B peptide-based immunoassays in the serodiagnosis of HCV infection in Cameroon subjects. STUDY DESIGN: Three core and four NS4B-based synthetic peptides derived from HCV genotypes 1b and 2a were designed and tested against a panel of 151 serum samples from Cameroon (40 positive for HCV-1, 32 for HCV-2, 39 HCV-4, and 40 HCV-negative). RESULTS: The three core peptides all demonstrated strong immunoreactivity, regardless of the HCV genotype from which they were derived, with greater than 90% and 92% sensitivity and specificity. In contrast, the NS4B-derived peptides exhibited lower sensitivities (24.3-65.8% depending on the HCV genotype) but higher specificities (100% for all four peptides tested). CONCLUSIONS: Our findings indicate that an HCV core peptide could be used for the diagnosis of chronic HCV infection. Among the NS4B peptides tested, a chimeric NS4B peptide encompassing both N- and C-terminal portions of the NS4B protein gave a much better performance than the two component N- and C-terminal peptides used individually