Functional selection of hepatitis C virus envelope E2-binding Peptide ligands by using ribosome display.

Silencing of USP18 Potentiates the Antiviral Activity of Interferon Against Hepatitis C Virus Infection

BackgroundHepatic steatosis is a risk factor for both liver disease progression and an impaired response to interferon alpha (IFN-α)-based combination therapy in chronic hepatitis C virus (HCV) infection. Previously, we reported that free fatty acid (FFA)-treated HCV cell culture induces hepatocellular steatosis and impairs the expression of interferon alpha receptor-1 (IFNAR1), which is why the antiviral activity of IFN-α against HCV is impaired.AimTo investigate the molecular mechanism by which IFNAR1 expression is impaired in HCV cell culture with or without free fatty acid-treatment.MethodHCV-infected Huh 7.5 cells were cultured with or without a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in HCV-infected culture was visualized by oil red staining. Clearance of HCV in FFA cell culture treated with type I IFN (IFN-α) and Type III IFN (IFN-λ) was determined by Renilla luciferase activity, and the expression of HCV core was determined by immunostaining. Activation of Jak-Stat signaling in the FFA-treated HCV culture by IFN-α alone and IFN-λ alone was examined by Western blot analysis and confocal microscopy. Lysosomal degradation of IFNAR1 by chaperone-mediated autophagy (CMA) in the FFA-treated HCV cell culture model was investigated.ResultsFFA treatment induced dose-dependent hepatocellular steatosis and lipid droplet accumulation in HCV-infected Huh-7.5 cells. FFA treatment of infected culture increased HCV replication in a concentration-dependent manner. Intracellular lipid accumulation led to reduced Stat phosphorylation and nuclear translocation, causing an impaired IFN-α antiviral response and HCV clearance. Type III IFN (IFN-λ), which binds to a separate receptor, induces Stat phosphorylation, and nuclear translocation as well as antiviral clearance in FFA-treated HCV cell culture. We show here that the HCV-induced autophagy response is increased in FFA-treated cell culture. Pharmacological inhibitors of lysosomal degradation, such as ammonium chloride and bafilomycin, prevented IFNAR1 degradation in FFA-treated HCV cell culture. Activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased IFNAR1 levels in Huh-7.5 cells. Co-immunoprecipitation, colocalization and siRNA knockdown experiments revealed that IFNAR1 but not IFNLR1 interacts with HSC70 and LAMP2A, which are core components of chaperone-mediated autophagy (CMA).ConclusionOur study presents evidence indicating that chaperone-mediated autophagy targets IFNAR1 degradation in the lysosome in FFA-treated HCV cell culture. These results provide a mechanism for why HCV induced autophagy response selectively degrades type I but not the type III IFNAR1.

A monocytic detour to replicate patient-derived hepatitis C virus in hepatoma cells and its use for phenotypic analyses.

Plant or microbial lectins are known to exhibit potent antiviral activities against viruses with glycosylated surface proteins, yet the mechanism(s) by which these carbohydrate-binding proteins exert their antiviral activities is not fully understood. Hepatitis C virus (HCV) is known to possess glycosylated envelope proteins (gpE1E2) and to be potently inhibited by lectins. Here, we tested in detail the antiviral properties of the newly discovered Microcystis viridis lectin (MVL) along with cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA) against cell culture HCV, as well as their binding properties toward viral particles, target cells, and recombinant HCV glycoproteins. Using infectivity assays, CV-N, MVL, and GNA inhibited HCV with IC50 values of 0.6 nM, 30.4 nM, and 11.1 nM, respectively. Biolayer interferometry analysis demonstrated a higher affinity of GNA to immobilized recombinant HCV glycoproteins compared to CV-N and MVL. Complementary studies, including fluorescence-activated cell sorting (FACS) analysis, confocal microscopy, and pre- and post-virus binding assays, showed a complex mechanism of inhibition for CV-N and MVL that includes both viral and cell association, while GNA functions by binding directly to the viral particle. Combinations of GNA with CV-N or MVL in HCV infection studies revealed synergistic inhibitory effects, which can be explained by different glycan recognition profiles of the mainly high-mannoside specific lectins, and supports the hypothesis that these lectins inhibit through different and complex modes of action. Our findings provide important insights into the mechanisms by which lectins inhibit HCV infection. Overall, the data suggest MVL and CV-N have the potential for toxicity due to interactions with cellular proteins while GNA may be a better therapeutic agent due to specificity for the HCV gpE1E2.

For many years, the standard of treatment for hepatitis C virus (HCV) infection was a combination of pegylated interferon alpha (Peg-IFN-α) and ribavirin for 24–48 weeks. This treatment regimen results in a sustained virologic response (SVR) rate in about 50 % of cases. The failure of IFN-α-based therapy to eliminate HCV is a result of multiple factors including a suboptimal treatment regimen, severity of HCV-related diseases, host factors and viral factors. In recent years, advances in HCV cell culture have contributed to a better understanding of the viral life cycle, which has led to the development of a number of direct-acting antiviral agents (DAAs) that target specific key components of viral replication, such as HCV NS3/4A, HCV NS5A, and HCV NS5B proteins. To date, several new drugs have been approved for the treatment of HCV infection. Application of DAAs with IFN-based or IFN-free regimens has increased the SVR rate up to >90 % and has allowed treatment duration to be shortened to 12–24 weeks. The impact of HCV proteins in response to IFN-based and IFN-free therapies has been described in many reports. This review summarizes and updates knowledge on molecular mechanisms of HCV proteins involved in anti-IFN activity as well as examining amino acid variations and mutations in several regions of HCV proteins associated with the response to IFN-based therapy and pattern of resistance associated amino acid variants (RAV) to antiviral agents.

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. We recently characterized for the first time the expression of Signaling Lymphocyte Activating Molecule 3 (SLAMF3) in human hepatocytes and here, we report that SLAMF3 interacts with the HCV viral protein E2 and is implicated in HCV entry process. We found a strong correlation between SLAMF3 expression level and hepatocyte susceptibility to HCV infection. The use of specific siRNAs to down-modulate SLAMF3 expression and SLAMF3-blocking antibodies both decreased the hepatocytes susceptibility to HCV infection. Moreover, SLAMF3 over-expression significantly increased susceptibility to HCV infection. Interestingly, experiments with peptides derived from each SLAMF3 domain showed that the first N-terminal extracellular domain is essential for interaction with HCV particles. Finally, we showed that recombinant HCV envelop protein E2 can bind SLAMF3 and that anti-SLAMF3 antibodies inhibited specifically this interaction. Overall, our results revealed that SLAMF3 plays a role during HCV entry, likely by enhancing entry of viral particle within hepatocytes.

Currently, no vaccine to prevent hepatitis C virus (HCV) infection is available. A major challenge in developing an HCV vaccine is the high diversity of HCV sequences. The purpose of immunization with viral glycoproteins is to induce a potent and long-lasting cellular and humoral immune response. However, this strategy only achieves limited protection, and antigen selection plays a crucial role in vaccine design. In this study, we investigated the humoral immune responses induced by intraperitoneal injection of keyhole limpet hemocyanin conjugated with 4 highly conserved peptides, including amino acids [aa]317-325 from E1 and aa418-429, aa502-518, and aa685-693 from E2, or 3 peptides from hypervariable region 1 (HVR1) of E2, including the N terminus of HVR1 (N-HVR1, aa384-396), C terminus of HVR1 (C-HVR1, aa397-410), and HVR1 in BALB/c mice. The neutralizing activity against HCV genotypes 1-6 was assessed using the cell culture HCV (HCVcc) system. The results showed that the 4 conserved peptides efficiently induced antibodies with potent neutralizing activity against 3 or 4 genotypes. Antibodies induced by aa685-693 conferred potent protection (>50%) against genotypes 2, 4, and 5. Peptide N-HVR1 elicited antibodies with the most potent neutralization activities against 3 HCV genotypes: TNcc(1a), S52(3a), and ED43(4a). These findings suggested that peptides within HCV glycoproteins could serve as potent immunogens for vaccine design and development.

Several enveloped viruses including human immunodeficiency virus type 1 (HIV-1), cytomegalovirus (CMV), herpes simplex virus 1 (HSV-1), Ebola virus, vaccinia virus, and influenza virus have been found to incorporate host regulators of complement activation (RCA) into their viral envelopes and, as a result, escape antibody-dependent complement-mediated lysis (ADCML). Hepatitis C virus (HCV) is an enveloped virus of the family Flaviviridae and incorporates more than 10 host lipoproteins. Patients chronically infected with HCV develop high-titer and crossreactive neutralizing antibodies (nAbs), yet fail to clear the virus, raising the possibility that HCV may also use the similar strategy of RCA incorporation to escape ADCML. The current study was therefore undertaken to determine whether HCV virions incorporate biologically functional CD59, a key member of RCA. Our experiments provided several lines of evidence demonstrating that CD59 was associated with the external membrane of HCV particles derived from either Huh7.5.1 cells or plasma samples from HCV-infected patients. First, HCV particles were captured by CD59-specific Abs. Second, CD59 was detected in purified HCV particles by immunoblot analysis and in the cell-free supernatant from HCV-infected Huh7.5.1 cells, but not from uninfected or adenovirus serotype 5 (Ad5) (a nonenveloped cytolytic virus)-infected Huh7.5.1 cells by enzyme-linked immunosorbent assay. Last, abrogation of CD59 function with its blockers increased the sensitivity of HCV virions to ADCML, resulting in a significant reduction of HCV infectivity. Additionally, direct addition of CD59 blockers into plasma samples from HCV-infected patients increased autologous virolysis. Our study, for the first time, demonstrates that CD59 is incorporated into both cell line-derived and plasma primary HCV virions at levels that protect against ADCML. This is also the first report to show that direct addition of RCA blockers into plasma from HCV-infected patients renders endogenous plasma virions sensitive to ADCML.

Hepatitis C virus (HCV) is a unique virus whose life cycle is closely associated with lipoprotein metabolism [1, 2, 3]. Assembly of HCV particles, formation of HCV-virions, is closely connected to the formation of lipid droplets in hepatic cells that may serve as an assembly platform [1, 4]. In addition, the production of HCV particles is tightly linked to the very low-density lipoprotein (VLDL) production pathway [5, 6]. HCV particles circulating in the blood during chronic HCV infection form lipo-viral particles (LVP) that are rich in triglycerides (TG), apoB-100 and apoE, with physiochemical similarity to VLDL particles and are highly infectious [7, 8]. In contrast, denser HCV particles are less infectious. These data strongly suggest that both viral particles and VLDL are integral components of LVPs with high infective capability. Although LVPs are thought to be assembled in liver cells by association with host lipoproteins prior to secretion, association between HCV and VLDL in the circulation after secretion from the liver cannot be ruled out.

An immunosuppressive function of interleukin-35 in chronic hepatitis C virus infection

Little is known about the ability of hepatitis C virus (HCV) to alter early innate immune responses in infected patients. Previous studies have shown that natural killer (NK) cells are functionally impaired after interaction of recombinant HCV glycoprotein E2 with the co-stimulatory CD81 molecule in vitro; however, the functional consequences of a prolonged contact of NK cells with HCV particles have remained unclear. We have examined the phenotypes of purified, interleukin-2-activated NK cells from healthy donors and HCV genotype 1b patients after culture for 5 days with HCV pseudoparticles (HCVpp) and serum samples containing HCV genotype 1b. NK cells from healthy donors and chronic HCV patients were found to up-regulate receptors associated with activation (NKp46, NKp44, NKp30, NKG2D), while NK receptors from the killer cell immunoglobulin-like receptor family (KIR/CD158), predominantly having an inhibitory function, were significantly down-modulated after culture in the presence of HCV particles compared with control cultures of NK cells. HCV-infected sera and HCVpp elicited significantly higher secretion of the NK effector lymphokines interferon-γ and tumour necrosis factor-α. Furthermore, HCV stimulated the cytotoxic potential of NK cells from normal donors and patients. The enhanced activation of NK cells after prolonged culture with HCVpp or HCV-containing sera for 5 days suggests that these innate effector cells may play an important role in viral control during early phases of HCV infection.

Although much has been learned about Hepatitis C virus (HCV), research progress has been hindered by the lack of a suitable cell culture system supporting its replication. Recently, a unique HCV strain JFH1 has been found to replicate efficiently in cell culture with production of infectious HCV (HCVcc). Baculovirus vectors were found to be efficient delivery vehicles and a HBV recombinant baculovirus/HepG2 system efficiently delivered the HBV genome into HepG2 resulting in HBV replication. In this study, we developed a recombinant baculovirus expression system to generate infectious HCV particles in hepatoma cell line Huh7-lunetT7 by using cDNA from the HCV JFH1 genotype. Results show that HCV positive, negative RNA strands and proteins were produced in this system. Furthermore, HCV particles were produced and secreted into the culture medium. Sucrose density gradient centrifugation of the culture medium revealed co-localization of HCV RNA and structural proteins in the fraction with a density of 1.08-1.13 g/ml. Electron microscopy (EM) showed viral particles approximately 55 nm in diameter, which could be recognized by anti-HCV E2 antibodies. Real-time RT-PCR detected that the level of HCV vRNA in the supernatant was 10(7) copies/ml at 72 h post-transduction (hpt). In addition, the JFH1 virus produced by the recombinant baculovirus was confirmed to be infectious in vitro. In summary, this system provides a novel tool not only for the analysis of the replication and pathogenesis of HCV but also to screen for potential therapeutic targets.

P.175 Interaction of serum-derived hepatitis C virus particles (HCVsp) and human hepatocytes: inhibition of binding by anti-E1E2 antibodies

Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteins trans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, these trans-complemented HCV particles (HCV(TCP)) penetrate target cells in a CD81 receptor-dependent fashion. Since HCV(TCP) production was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCV(TCP) stocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 10(6) tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excluding cis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCV(TCP) may prove valuable for gene delivery or vaccination approaches.

We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.