The liver-specific microRNA miR-122 is required for efficient hepatitis C virus (HCV) RNA replication both in cell culture and in vivo. In addition, nonhepatic cells have been rendered more efficient at supporting this stage of the HCV life cycle by miR-122 expression. This study investigated how miR-122 influences HCV replication in the miR-122-deficient HepG2 cell line. Expression of this microRNA in HepG2 cells permitted efficient HCV RNA replication and infectious virion production. When a missing HCV receptor is also expressed, these cells efficiently support viral entry and thus the entire HCV life cycle.Hepatitis C virus (HCV), a member of the family Flaviviridae, is associated with more than half of newly diagnosed hepatocellular carcinomas in the United States (1, 31) and is the leading cause of liver transplants worldwide (7). Historically, a lack of model systems to study HCV replication has slowed the progress of HCV research and the development of specific antivirals. Few HCV genomes replicate in culture, and only one efficiently produces infectious particles (23,39,42). Most model systems rely on the Huh-7 cell line and derivatives, such as the highly HCV-permissive Huh-7.5 subclone (6). The development of novel cell systems capable of supporting the entire HCV life cycle would allow broader examination of interactions between HCV and host cell biology and would provide essential tools for anti-HCV drug discovery.MicroRNAs (miRNAs) are small RNA molecules predicted to downregulate the expression of one-third of human genes by reducing mRNA stability and/or translation, depending on the degree of complementarity between the miRNA and the mRNA target site (2). miR-122, a liver-specific miRNA expressed at high levels in hepatocytes, is required to support HCV RNA replication (19). Two miR-122 target sites have been identified in the 5Ј untranslated region of the HCV genome, and mutation of these sites disrupts HCV RNA replication. Replication is restored by cotransfection with RNA duplexes that produce a mutant miR-122 with complementary changes (16)(17)(18)(19)26). How miR-122 enhances HCV replication is unclear. Although its binding appears to enhance HCV internal ribosome entry site (IRES)-directed translation, this effect is not great enough to account for the difference in RNA replication (16,17,40). Furthermore, the normal cellular function of miR-122 in regulating cholesterol and fatty acid biosynthesis is not required for this enhancement (18). Thus, miR-122 has been hypothesized to directly affect HCV RNA replication or stability (17,19,26). Although inhibiting miR-122 has been shown to be an effective therapy in chimpanzees infected with HCV (21), further development of such therapies will require a firmer understanding of how miR-122 promotes HCV replication.The majority of published studies on the role of miR-122 in the HCV life cycle have described research conducted with HCV-permissive Huh-7 cells or derivatives, which express endogenous miR-122 (19). Two studies explored the effect of m...

Hepatitis C virus (HCV) exposure leads to persistent life-long infections characterized by chronic inflammation often developing into cirrhosis and hepatocellular carcinoma. The mechanism by which HCV remains in the liver while inducing an inflammatory and antiviral response remains unclear. While the innate immune response to HCV in patients seem to be quite active, HCV has been shown in cell culture to employ a diverse array of innate immune antagonists, suggesting that current model systems to study interactions between HCV and the innate immune system are not representative of what is happening in vivo. We recently showed that hepatoma-derived HepG2 cells support the entire HCV life cycle if the liver-specific microRNA miR-122 is expressed along with the entry factor CD81 (HepG2-HFL cells). We found that there was a striking difference in these cells’ ability to sustain HCV infection and spread when compared to Huh-7 and Huh-7.5 cells. Additionally, HepG2-HFL cells produced a more robust antiviral response when challenged with other RNA viruses and viral mimetics than Huh-7 and Huh-7.5 cells. HCV infection elicited a potent IFN-λ (lambda), ISG, and cytokine response in HepG2-HFL cells, but not in Huh-7 cells, suggesting that HepG2-HFL cells more faithfully recapitulate the innate immune response to HCV infection in vivo. Using this new model, we found that blocking the RIG-I like receptor pathway or the IFN-λ signaling pathway, promoted HCV infection and spread in HepG2-HFL cells. Conclusion HepG2-HFL cells represent a promising new system to study the interaction between HCV and the innate immune system, solidifying the importance of IFN-λ in hepatic response to HCV infection and revealing non-redundant roles of RIG-I and MDA5 in HCV recognition and repression of infection.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. A better understanding of its life cycle, including the process of host cell entry, is important for the development of HCV therapies and model systems. Based on the requirement for numerous host factors, including the two tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), HCV cell entry has been proposed to be a multi-step process. The lack of OCLN-specific inhibitors has prevented a comprehensive analysis of this process. To study the role of OCLN in HCV cell entry, we created OCLN mutants whose HCV cell entry activities could be inhibited by antibodies. These mutants were expressed in polarized HepG2 cells engineered to support the complete HCV life cycle by CD81 and miR-122 expression and synchronized infection assays were performed to define the kinetics of HCV cell entry. During these studies, OCLN utilization differences between HCV isolates were observed, supporting a model that HCV directly interacts with OCLN. In HepG2 cells, both HCV cell entry and tight junction formation were impaired by OCLN silencing and restored by expression of antibody regulatable OCLN mutant. Synchronized infection assays showed that glycosaminoglycans and SR-BI mediated host cell binding, while CD81, CLDN1 and OCLN all acted sequentially at a post-binding stage prior to endosomal acidification. These results fit a model where the tight junction region is the last to be encountered by the virion prior to internalization.

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. As HCV infects only human and chimpanzee cells, antiviral therapy and vaccine development have been hampered by the lack of a convenient small-animal model. In this study we further investigate how the species tropism of HCV is modulated at the level of cell entry. It has been previously determined that the tight junction protein occludin (OCLN) is essential for HCV host cell entry and that human OCLN is more efficient than the mouse ortholog at mediating HCV cell entry. To further investigate the relationship between OCLN sequence and HCV species tropism, we compared OCLN proteins from a range of species for their ability to mediate infection of naturally OCLNdeficient 786-O cells with lentiviral pseudoparticles bearing the HCV glycoproteins. While primate sequences function equivalently to human OCLN, canine, hamster, and rat OCLN had intermediate activities, and guinea pig OCLN was completely nonfunctional. Through analysis of chimeras between these OCLN proteins and alanine scanning mutagenesis of the extracellular domains of OCLN, we identified the second half of the second extracellular loop (EC2) and specific amino acids within this domain to be critical for modulating the HCV cell entry factor activity of this protein. Furthermore, this critical region of EC2 is flanked by two conserved cysteine residues that are essential for HCV cell entry, suggesting that a subdomain of EC2 may be defined by a disulfide bond.Hepatitis C virus (HCV), a member of the family Flaviviridae, is the causative agent of classically defined non-A, non-B hepatitis and is highly prevalent, with approximately 3% of the worldwide population infected (48). HCV infection often results in a chronic, life-long infection that can have severe health consequences, including hepatitis, cirrhosis, hepatocellular carcinoma, and liver failure. There is no HCV vaccine available, and the currently employed interferon-based treatment is inadequate as it has severe side effects and is effective only in half of the major genotype-infected individuals (22,32). Specific anti-HCV inhibitors targeting the viral proteases and polymerase are currently being developed and will likely improve therapeutic options substantially. Undoubtedly, however, the emergence of viral resistance to such inhibitors will be a problem facing future HCV treatment options. As such, developing a spectrum of inhibitors targeting diverse steps in the virus life cycle, including HCV cell entry, is a priority for HCV research. Such inhibitors may be particularly useful following liver transplantation. Although HCV is the leading cause of liver transplants worldwide (10), the usefulness of such procedures is limited by subsequent universal graft reinfection and often accelerated disease progression (21). Even transiently inhibiting graft reinfection with HCV cell entry inhibitors could greatly improve the effectiveness of this procedure. Therefore, a greater understanding of HCV cell entry is required for the ...