Hepatitis C virus (HCV) circulates in the bloodstream in different forms, including complexes with immunoglobulins and/or lipoproteins.To address the significance of such associations, we produced or treated HCV pseudoparticles (HCVpp), a valid model of HCV cell entry and its inhibition, with naïve or patient-derived sera. We demonstrate that infection of hepatocarcinoma cells by HCVpp is increased more than 10-fold by human serum factors, of which high-density lipoprotein (HDL) is a major component. Infection enhancement requires scavenger receptor BI, a molecule known to mediate HDL uptake into cells as well as HCVpp entry, and involves conserved amino acid positions in hypervariable region 1 (HVR1) of the E2 glycoprotein. Additionally, we show that the interaction with human serum or HDL, but not with low-density lipoprotein, leads to the protection of HCVpp from neutralizing antibodies, including monoclonal antibodies and antibodies present in patient sera. Finally, the deletion or mutation of HVR1 in HCVpp abolishes infection enhancement and leads to increased sensitivity to neutralizing antibodies/sera compared to that of parental HCVpp. Altogether, these results assign to HVR1 new roles which are complementary in helping HCV to survive within its host. Besides immune escape by mutation, HRV1 can mediate the enhancement of cell entry and the protection of virions from neutralizing antibodies. By preserving a balance between these functions, HVR1 may be essential for the viral persistence of HCV.Hepatitis C poses a major public health problem, with nearly 3% of the world's population infected and approximately 3 to 4 million new infections occurring each year (22). Hepatitis C virus (HCV) infection has become very prevalent, with about 5 million cases in Europe, 4 million in the United States, and 2 million in Japan. In the United States, HCV infection is the most common chronic blood-borne infection, and HCV-associated chronic liver disease is the principal cause of liver transplantation and the 10th leading cause of death among adults (27). At present, there exists no vaccine against HCV infection, and the only authorized treatments, pegylated alpha interferon and ribavirin, have shown limited effects against HCV, with sustained virological response rates of 54% in general and 42% for genotype 1. Furthermore, the treatments cause significant side effects (28).HCV is transmitted by blood and progresses slowly, causing no symptoms or only mild symptoms in the acute phase of infection. However, only 20% of infected individuals clear the virus spontaneously, while 80% develop chronic disease which leads to various severe hepatic pathologies (cirrhosis and hepatocarcinoma) in the long term in one out of five cases. Spontaneous clearance of HCV has been associated with strong cellular immune responses (reviewed in reference 48), while detailed analysis of the role of the humoral immune response has become possible only recently with the development of HCV pseudoparticles (HCVpp), a recently described model of H...

Because of the lack of a robust cell culture system, relatively little is known about the molecular details of the cell entry mechanism for hepatitis C virus (HCV). Recently, we described infectious HCV pseudo-particles (HCVpp) that were generated by incorporating unmodified HCV E1E2 glycoproteins into the membrane of retroviral core particles. These initial studies, performed with E1E2 glycoproteins of genotype 1, noted that HCVpp closely mimic the cell entry and neutralization properties of parental HCV. Because sequence variations in E1 and E2 may account for differences in tropism, replication properties, neutralization, and response to treatment in patients infected with different genotypes, we investigated the functional properties of HCV envelope glycoproteins from different genotypes/subtypes. Our studies indicate that hepatocytes were preferential targets of infection in vitro, although HCV replication in extrahepatic sites has been reported in vivo. Receptor competition assays using antibodies against the CD81 ectodomain as well as ectopic expression of CD81 in CD81-deficient HepG2 cells indicated that CD81 is used by all the different genotypes/subtypes analyzed to enter the cells. However, by silencing RNA (siRNA) interference assays, our results show that the level of Scavenger Receptor Class-B Type-I (SR-BI) needed for efficient infection varies between genotypes and subtypes. Finally, sera from chronic HCV carriers were found to exhibit broadly reactive activities that inhibited HCVpp cell entry, but failed to neutralize all the different genotypes. In conclusion, we characterize common steps in the cell entry pathways of the major HCV genotypes that should provide clues for the development of cell entry inhibitors and vaccines. (HEPATOLOGY 2005;41:265-274.) H epatitis C virus (HCV) is an RNA-enveloped virus belonging to the Flaviviridae family. HCV exhibits a high degree of genetic heterogeneity. The propensity for genetic change is associated primarily with the error-prone nature of its RNA-dependent RNA polymerase together with the high HCV replicative rate in vivo. 1,2 This results in infected individuals harboring a diverse population of viral variants known as a quasispecies, which evolve in response to a variety of selective pressures. 3 Although HCVspecific immunity develops after primary infection, it frequently fails to eliminate the virus. 4-6 HCV has infected approximately 170 million people worldwide, and approximately 80% of those infected will develop chronic infection. The outcome of chronic infection varies widely between individuals, but large proportions of patients develop serious liver diseases such as cirrhosis and hepatocellular carcinoma. 7 Current therapies are inadequate, and development of appropriate therapeutic and prophylactic vaccines remains a significant challenge.HCV can be classified into six genetically distinct genotypes and further subdivided into at least 70 subtypes, which differ by approximately 30% and 15% at the nu-

The factors leading to spontaneous clearance of hepatitis C virus (HCV) or to viral persistence are elusive. Understanding virus-host interactions that enable acute HCV clearance is key to the development of more effective therapeutic and prophylactic strategies. Here, using a sensitive neutralization assay based on infectious HCV pseudoparticles (HCVpp), we have studied the kinetics of humoral responses in a cohort of acute-phase patients infected during a single nosocomial outbreak in a hemodialysis center. The 17 patients were monitored for the spontaneous outcome of HCV infection for 6 months before a treatment decision was made. Blood samples were taken frequently (15 ؎ 4 per patient). Phylogenetic analysis of the predominant virus(es) revealed infection by only one of two genotype 1b strains. While all patients seroconverted, their sera induced two opposing effects in HCVpp infection assays: inhibition and facilitation. Furthermore, the ability of sera to facilitate or inhibit infection correlated with the presence of either infecting HCV strain and divided the patients into two groups. In group 1, the progressive emergence of a relatively strong neutralizing response correlated with a fluctuating decrease in high initial viremia, leading to control of viral replication. Patients in group 2 failed to reduce viremia within the acute phase, and no neutralizing responses were detected despite seroconversion. Strikingly, sera of group 2, as well as naïve sera, facilitated infection by HCVpp displaying HCV glycoproteins from different genotypes and strains, including those retrieved from patients. These results provide new insights into the mechanisms of viral persistence and immune control of viremia.Hepatitis C virus (HCV) infection causes acute hepatitis after 4 to 12 weeks of incubation. Acute hepatitis is characterized by elevated alanine aminotransferase (ALT) levels, with generally no or only mild symptoms. Among infected individuals, only 20% clear infection spontaneously, whereas ϳ80% progress to chronic infection. Chronic hepatitis may lead after 10 to 30 years to severe, life-threatening complications, such as cirrhosis and hepatocellular carcinoma. With an estimated 170 million people infected, i.e., nearly 3% of the world population, and an incidence of ϳ3 to 4 million new infections per year, HCV is presently a leading cause of chronic liver disease and poses a major public health problem. In the United States, HCV constitutes the most common chronic blood-borne infectious disease and is the principal indication for liver transplantation and the 10th leading cause of deaths among adults. The only approved therapy for chronic hepatitis C is the combination of alpha interferon, used in a pegylated form, and ribavirin. This treatment cures infection in a significant proportion of patients, but its efficacy against HCV genotype 1, the most frequent HCV genotype in industrialized countries, remains limited, and it can cause significant side effects (18,23,36). HCV is a highly variable virus that compri...