Hepatitis C virus-like particles (HCV-LPsHepatitis C virus (HCV) is the major etiology of non-A, non-B hepatitis that infects 170 million people worldwide. Approximately 70 to 80% of HCV patients develop chronic hepatitis, 20 to 30% of which progress to liver cirrhosis (52). At present, there is no vaccine available to prevent HCV infection, and current therapies are not optimal. The initial steps of HCV infection (binding and entry) that are critical for tissue tropism, and hence pathogenesis, are poorly understood. Studies to elucidate this process have been hampered by the lack of robust cell culture systems or convenient small animal models that can support HCV infection.HCV is an enveloped, positive-strand RNA virus that belongs to the Flaviviridae family. Based on the sequence heterogeneity of the genome, HCV is classified into six major genotypes and ϳ100 subtypes (52). The viral genome (ϳ9.6 kb) is translated into a single polyprotein of ϳ3,000 amino acids (aa). A combination of host and viral proteases are involved in polyprotein processing to give at least nine different proteins (for a review, see reference 4). The structural proteins of HCV are believed to comprise the core protein (ϳ21 kDa) and two envelope glycoproteins: E1 (ϳ31 kDa) and E2 (ϳ70 kDa). Like other enveloped viruses, E1 and E2 proteins most likely play a pivotal role in HCV life cycle: in the assembly of infectious particle and in the initiation of viral infection by binding to its cellular receptor(s). Since hepatocytes represent the primary site of HCV replication in vivo, the HCV genome has also been found in lymphoid cells. Infection of the lymphoid cells has been implicated in extrahepatic manifestations of HCV infection such as mixed cryoglobulinemia and B-lymphocyte proliferative disorders (2, 39, 42).Detail ultrastructural features of the HCV virion remain elusive since direct visualization of virus particles from infected serum and tissues has proven to be difficult. Previous studies have shown that HCV particles vary in size between 30 and 60 nm in diameter (24,38,43). In addition, HCV particles display significant heterogeneity in buoyant density on sucrose density gradient centrifugation, ranging from low (Ͻ1.07 g/ml) to high (1.25 g/ml) density (22,24,47,54). The heterogeneity of the particle density has been attributed to the variability in size (44), nonenveloped nucleocapsid particles (28,48), and an association with antibodies or -lipoproteins (38, 47).To date, the cellular receptor(s) for HCV remains controversial. The observations that HCV can infect both hepatic and lymphoid cells suggest that HCV may use different cellular receptors to access different cell types. However, the absence of an in vitro system that supports HCV replication and particle assembly has hampered studies to elucidate the early steps of HCV infection, i.e., virus binding and entry. Association of HCV virions with -lipoproteins in plasma has raised the possibility that HCV may use low-density lipoprotein receptor (LDL-R) for viral entry (1,...
We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.Hepatitis C virus (HCV) infection has become a major health problem affecting an estimated 170 million people worldwide. Persistent infection occurs in more than 70% of people infected with HCV, which may be complicated by cirrhosis and/or hepatocellular carcinoma (24, 33). Despite highly competitive and extensive research in this field, a highly effective treatment is not yet available. The mainstay of anti-HCV therapy, alpha interferon (IFN-␣) or pegylated IFN-␣, together with ribavirin leads, at best, to viral clearance in ca. 41 to 54% of patients infected with HCV genotype 1a (34,38). Since mechanisms of HCV infection remain unclear, characterization of these mechanisms is now a major issue for the development of new strategies for anti-HCV treatment and prevention.
ABSTRACT.Early prediction of human clearance is often challenging, in particular for the growing number of low-clearance compounds. Long-term in vitro models have been developed which enable sophisticated hepatic drug disposition studies and improved clearance predictions. Here, the cell line HepG2, iPSC-derived hepatocytes (iCell®), the hepatic stem cell line HepaRG™, and human hepatocyte co-cultures (HμREL™ and HepatoPac®) were compared to primary hepatocyte suspension cultures with respect to their key metabolic activities. Similar metabolic activities were found for the long-term models HepaRG™, HμREL™, and HepatoPac® and the short-term suspension cultures when averaged across all 11 enzyme markers, although differences were seen in the activities of CYP2D6 and non-CYP enzymes. For iCell® and HepG2, the metabolic activity was more than tenfold lower. The micropatterned HepatoPac® model was further evaluated with respect to clearance prediction. To assess the in vitro parameters, pharmacokinetic modeling was applied. The determination of intrinsic clearance by nonlinear mixed-effects modeling in a long-term model significantly increased the confidence in the parameter estimation and extended the sensitive range towards 3% of liver blood flow, i.e., >10-fold lower as compared to suspension cultures. For in vitro to in vivo extrapolation, the well-stirred model was used. The micropatterned model gave rise to clearance prediction in man within a twofold error for the majority of low-clearance compounds. Further research is needed to understand whether transporter activity and drug metabolism by non-CYP enzymes, such as UGTs, SULTs, AO, and FMO, is comparable to the in vivo situation in these long-term culture models.KEY WORDS: in vitro clearance; in vitro liver models; IVIVE; nonlinear mixed-effects modeling.
The envelope glycoprotein E2 of hepatitis C virus (HCV) is a major component of the viral envelope. Knowledge of its topologic features and antigenic determinants in virions is crucial in understanding the viral binding sites to cellular receptor(s) and the induction of neutralizing antibodies. The lack of a robust cell culture system for virus propagation has hampered the characterization of E2 presented on the virion. Here we report the structural features of hepatitis C virus-like particles (HCV-LPs) of the 1a and 1b genotypes as determined by various mouse and human monoclonal anti-envelope antibodies. Our results show that the E2 protein of HCV-LPs reacts with human monoclonal antibodies recognizing conformational determinants. Monoclonal antibodies (mAbs) specific for the hypervariable region 1 (HVR-1) sequence reacted strongly with HCV-LPs, suggesting that the HVR-1 is exposed on the viral surface. Several mAbs recognized both HCV-LPs with equally high affinity, indicating that the corresponding epitopes [amino acids (aa) 192-217 of E1 and aa 412-423, aa 522-531, and aa 640-653 of E2] are conserved in both genotypes and exposed on the surface of the HCV-LP. The E2 and E1/E2 dimers of 1a bound strongly to the recombinant large extracellular loop (LEL) of CD81 (CD81-LEL) of human and African green monkey, while the HCV-LP of 1a bound weakly to human CD81-LEL. E1/E2 dimers and the HCV-LPs of 1b did not bind CD81-LEL, consistent with the notion that CD81 recognition by E2 is strain-specific and does not correlate with permissiveness of infection. A model of the topology and exposed antigenic determinants of the envelope proteins of HCV is proposed.
Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8–12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.
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