Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with generally 4 and 11 N-linked glycans on E1 and E2, respectively. Studies using mutated recombinant HCV envelope glycoproteins incorporated into retroviral pseudoparticles (HCVpp) suggest that some glycans play a role in protein folding, virus entry, and protection against neutralization. The development of a cell culture system producing infectious particles (HCVcc) in hepatoma cells provides an opportunity to characterize the role of these glycans in the context of authentic infectious virions. Here, we used HCVcc in which point mutations were engineered at N-linked glycosylation sites to determine the role of these glycans in the functions of HCV envelope proteins. The mutants were characterized for their effects on virus replication and envelope protein expression as well as on viral particle secretion, infectivity, and sensitivity to neutralizing antibodies. Our results indicate that several glycans play an important role in HCVcc assembly and/or infectivity. Furthermore, our data demonstrate that at least five glycans on E2 (denoted E2N1, E2N2, E2N4, E2N6, and E2N11) strongly reduce the sensitivity of HCVcc to antibody neutralization, with four of them surrounding the CD81 binding site. Altogether, these data indicate that the glycans associated with HCV envelope glycoproteins play roles at different steps of the viral life cycle. They also highlight differences in the effects of glycosylation mutations between the HCVpp and HCVcc systems. Furthermore, these carbohydrates form a "glycan shield" at the surface of the virion, which contributes to the evasion of HCV from the humoral immune response.
Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect Alpha interferon (IFN) is a Food and Drug Administrationapproved treatment for chronic HCV infection. Only 8 to 12% of patients with HCV genotype 1 have a sustained clinical virological response to IFN therapy (4,43,61). Recently, combination therapy with interferon and the guanosine analogue ribavirin was shown to be superior to IFN monotherapy in producing sustained biochemical and virological responses (9,45,62). However, despite the significant improvement in rates of sustained response, as many as 60% of patients with hightiter HCV genotype 1 infection are nonresponsive to combination therapy.When IFN binds to its receptor, two receptor-associated tyrosine kinases of the STAT/JAK family, Tyk2 and Jak1, become activated. These activated kinases phosphorylate STAT-1 and STAT-2 on a single conserved tyrosine residue (8). STAT-1 and STAT-2 form heterodimers and combine with the p48 protein to form an active transcription factor known as IFN-stimulated gene factor 3 (ISGF-3). ISGF-3 binds to a common element termed the interferon-stimulated response element (ISRE), found in the promoter regions of all IFNstimulated genes, whereupon transcription occurs. Expression of the entire HCV polyprotein has been shown to inhibit IFNinduced STAT/JAK signaling in human U2-OS osteosarcoma cells (25). It was not reported which HCV protein was responsible for this effect.Recent studies have led to exciting discoveries in the emerging research area of the roles of HCV proteins in antiviral resistance. Two examples are the interaction of the HCV non-* Corresponding author. Mailing address:
Hepatitis C is caused by an enveloped virus whose entry is mediated by two glycoproteins, namely, E1 and E2, which have been shown to assemble as a noncovalent heterodimer. Despite extensive research in the field of such an important human pathogen, hepatitis C virus (HCV) glycoproteins have only been studied so far in heterologous expression systems, and their organization at the surfaces of infectious virions has not yet been described. Here, we characterized the envelope glycoproteins associated with cell-cultured infectious virions and compared them with their prebudding counterparts. Viral particles were analyzed by ultracentrifugation, and the envelope glycoproteins were characterized by coimmunoprecipitation and receptor pulldown assays. Furthermore, their oligomeric state was determined by sedimentation through sucrose gradients and by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. In sucrose gradient analyses, HCV envelope glycoproteins were associated with fractions containing the most infectious viral particles. Importantly, besides maturation of some of their glycans, HCV envelope glycoproteins showed a dramatic change in their oligomeric state after incorporation into the viral particle. Indeed, virion-associated E1 and E2 envelope glycoproteins formed large covalent complexes stabilized by disulfide bridges, whereas the intracellular forms of these proteins assembled as noncovalent heterodimers. Furthermore, the virion-associated glycoprotein complexes were recognized by the large extracellular loop of CD81 as well as conformation-sensitive antibodies, indicating that these proteins are in a functional conformation. Overall, our study fills a gap in the description of HCV outer morphology and should guide further investigations into virus entry and assembly.Hepatitis C virus (HCV) infects 3% of the world population. It represents a major health burden due to its high propensity to cause chronic infections and the subsequent liver cirrhosis and carcinoma (for a review, see reference 31). Discovered in 1989, it is a small enveloped virus, classified into the Flaviviridae family. Its positive-strand RNA genome encodes a single polyprotein that is co-and posttranslationally processed into 10 mature proteins. From the N terminus to the C terminus of the polyprotein are located the precursors for three structural proteins, namely, the core protein (capsid protein) and E1 and E2 (envelope glycoproteins), and for seven nonstructural proteins (p7, NS2, 3, 4A, 4B, 5A, and 5B) (for a review, see reference 43).HCV circulates in infected patients at relatively low titers and forms complexes with serum components such as lipoproteins, antibodies or rheumatoid factors, and cryoglobulins (45). Furthermore, for reasons that remain partly unknown, serumderived HCV cannot be propagated in cell culture. HCV research has been hindered by these restrictions and has largely depended on surrogate systems. For instance, replication-deficient retroviruses pse...
Here, we identify (2)-epigallocatechin-3-gallate (EGCG) as a new inhibitor of hepatitis C virus (HCV) entry. EGCG is a flavonoid present in green tea extract belonging to the subclass of catechins, which has many properties. Particularly, EGCG possesses antiviral activity and impairs cellular lipid metabolism. Because of close links between HCV life cycle and lipid metabolism, we postulated that EGCG may interfere with HCV infection. We demonstrate that a concentration of 50 lM of EGCG inhibits HCV infectivity by more than 90% at an early step of the viral life cycle, most likely the entry step. This inhibition was not observed with other members of the Flaviviridae family tested. The antiviral activity of EGCG on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition, using binding assays at 48C, we demonstrate that EGCG prevents attachment of the virus to the cell surface, probably by acting directly on the particle. We also show that EGCG has no effect on viral replication and virion secretion. By inhibiting cell-free virus transmission using agarose or neutralizing antibodies, we show that EGCG inhibits HCV cell-to-cell spread. Finally, by successive inoculation of naïve cells with supernatant of HCV-infected cells in the presence of EGCG, we observed that EGCG leads to undetectable levels of infection after four passages. Conclusion: EGCG is a new, interesting anti-HCV molecule that could be used in combination with other direct-acting antivirals. Furthermore, it is a novel tool to further dissect the mechanisms of HCV entry into the hepatocyte. (HEPATOLOGY 2012;55:720-729) H epatitis C virus (HCV) is a major cause of chronic liver disease. It is estimated that 3% of the world population is currently infected and thus is at high risk of developing cirrhosis and hepatocellular carcinoma. 1 No vaccine is available, and the current standard-of-care therapy with pegylated interferon-alpha (IFN-a) and ribavirin has a limited efficacy and significant side effects. 2 Very recently, an addition to the therapy of new direct-acting antivirals (DAAs) targeting HCV nonstructural protein (NS)3-4A protease, telaprevir, and boceprevir was shown to increase the sustained virological response in patients infected with HCV genotype 1 by up to 70%. 3 Efforts are currently being made to identify new DAAs with additive potency. The majority of these molecules target the replication step. 3 However, because of the high genetic heterogeneity of HCV and its rapid replication, monotherapy with DAA agents poses a high risk for selection of resistant variants. 4 Therefore, combinations of drugs targeting different steps of the viral life cycle,
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