Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR-BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR-BI for productive HCV infection remains unclear. In this study, we investigated the role of SR-BI as an entry factor for infection of human hepatoma cells using cell culture–derived HCV (HCVcc). Anti–SR-BI antibodies directed against epitopes of the human SR-BI extracellular loop specifically inhibited HCVcc infection in a dose-dependent manner. Down-regulation of SR-BI expression by SR-BI–specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR-BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81–HCV interaction. Although the addition of high-density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti–SR-BI antibodies and SR-BI–specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR-BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV–CD81 interaction. (Hepatology 2007.)

The tight junction protein claudin‐1 (CLDN1) has been shown to be essential for hepatitis C virus (HCV) entry—the first step of viral infection. Due to the lack of neutralizing anti‐CLDN1 antibodies, the role of CLDN1 in the viral entry process is poorly understood. In this study, we produced antibodies directed against the human CLDN1 extracellular loops by genetic immunization and used these antibodies to investigate the mechanistic role of CLDN1 for HCV entry in an infectious HCV cell culture system and human hepatocytes. Antibodies specific for cell surface–expressed CLDN1 specifically inhibit HCV infection in a dose‐dependent manner. Antibodies specific for CLDN1, scavenger receptor B1, and CD81 show an additive neutralizing capacity compared with either agent used alone. Kinetic studies with anti‐CLDN1 and anti‐CD81 antibodies demonstrate that HCV interactions with both entry factors occur at a similar time in the internalization process. Anti‐CLDN1 antibodies inhibit the binding of envelope glycoprotein E2 to HCV permissive cell lines in the absence of detectable CLDN1‐E2 interaction. Using fluorescent‐labeled entry factors and fluorescence resonance energy transfer methodology, we demonstrate that anti‐CLDN1 antibodies inhibit CD81‐CLDN1 association. In contrast, CLDN1‐CLDN1 and CD81‐CD81 associations were not modulated. Taken together, our results demonstrate that antibodies targeting CLDN1 neutralize HCV infectivity by reducing E2 association with the cell surface and disrupting CD81‐CLDN1 interactions. Conclusion: These results further define the function of CLDN1 in the HCV entry process and highlight new antiviral strategies targeting E2‐CD81‐CLDN1 interactions. (HEPATOLOGY 2010.)

End-stage liver disease caused by chronic hepatitis C virus (HCV) infection is a leading cause for liver transplantation (LT). Due to viral evasion from host immune responses and the absence of preventive antiviral strategies, reinfection of the graft is universal. The mechanisms by which the virus evades host immunity to reinfect the liver graft are unknown. In a longitudinal analysis of six HCV-infected patients undergoing LT, we demonstrate that HCV variants reinfecting the liver graft were characterized by efficient entry and poor neutralization by antibodies present in pretransplant serum compared with variants not detected after transplantation. Monoclonal antibodies directed against HCV envelope glycoproteins or a cellular entry factor efficiently cross-neutralized infection of human hepatocytes by patient-derived viral isolates that were resistant to autologous host-neutralizing responses. These findings provide significant insights into the molecular mechanisms of viral evasion during HCV reinfection and suggest that viral entry is a viable target for prevention of HCV reinfection of the liver graft.

Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs. In spite of the rapid development of antiviral agents, antiviral resistance remains a challenge for the treatment of viral infections including hepatitis B and C virus (HBV, HCV), human immunodeficiency virus (HIV) and influenza. Virus spreads from infected cells to surrounding uninfected host cells to develop infection through cell-free and cell-cell transmission routes. Understanding the spread of resistant virus is important for the development of novel antiviral strategies to prevent and treat antiviral resistance. Here, we characterize the spread of resistant viruses and its impact for emergence and prevention of resistance using HCV as a model system. Our results show that cell-cell transmission is the main transmission route for antiviral resistant HCV strains and is crucial for the maintenance of infection. Monoclonal antibodies or small molecules targeting HCV entry factors are effective in inhibiting the spread of resistant HCV in cell culture models and thus should be evaluated clinically for prevention and treatment of HCV resistance. Combination of inhibitors targeting viral entry and clinically used direct-acting antivirals (DAAs) prevents antiviral resistance and leads to viral eradication in cell culture models. Collectively, the investigation provides a new strategy for prevention of viral resistance to antiviral agents.