The absence of a robust cell culture model of hepatitis C virus (HCV) infection has severely limited analysis of the HCV life cycle and the development of effective antivirals and vaccines. Here we report the establishment of a simple yet robust HCV cell culture infection system based on the HCV JFH-1 molecular clone and Huh-7-derived cell lines that allows the production of virus that can be efficiently propagated in tissue culture. This system provides a powerful tool for the analysis of host-virus interactions that should facilitate the discovery of antiviral drugs and vaccines for this important human pathogen.CD81 ͉ Huh-7 ͉ viral entry ͉ viral spread ͉ interferon H epatitis C virus (HCV) is a noncytopathic positive-stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma (1). The hepatocyte is the primary target cell, although various lymphoid populations, especially B cells and dendritic cells, may also be infected at lower levels (2-4). A striking feature of HCV infection is its tendency toward chronicity, with at least 70% of acute infections progressing to persistence (1), which is often associated with significant liver disease, including chronic active hepatitis, cirrhosis, and hepatocellular carcinoma (5). Thus, with Ͼ170 million people currently infected (5), HCV represents a growing public health burden.The HCV life cycle and host-virus interactions that determine the outcome of infection have been difficult to study, because cell culture and small animal models of HCV infection are not available. Thus, HCV infection studies to date have involved infected patients (6-8) and chimpanzees (9-12). The recent development of HCV replicon systems has also permitted the study of HCV translation and RNA replication in human hepatoma-derived Huh-7 cells in vitro (13,14), revealing some of the host-virus interactions that regulate these processes (15)(16)(17)(18)(19). Nonetheless, these replicons do not replicate efficiently without adaptive mutations (20, 21), nor do they produce infectious virions. Thus, the relevance of replicons to HCV infection is unclear, and they do not permit analysis of the complete viral life cycle.Wakita and colleagues (22, 23), however, have developed an HCV genotype 2a replicon (JFH-1) that replicates efficiently in Huh-7 cells, other human hepatocyte-derived cells (e.g., HepG2 and IMY-N9) (24), and nonhepatic cells (e.g., HeLa and HEK293) (25) without adaptive mutations. This group also recently reported that Huh-7 cells transfected with in vitro transcribed JFH-1 genomic RNA can secrete infectious viral particles. ʈ Unfortunately, the infection efficiency observed was low, and infectious particles could not be propagated in naïve ʈ).In contrast, we now report the establishment of a robust highly efficient in vitro infection system based on Huh-7-derived cell lines and the JFH-1 consensus clone. This system yields viral titers of 10 4 -10 5 infectious units per ml of culture supernatant; infection spreads throughout the culture within a few days a...

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Our laboratory has previously demonstrated that high-level HCV replication during acute infection of chimpanzees is associated with the modulation of multiple genes involved in lipid metabolism, and that drugs that regulate cholesterol and fatty acid biosynthesis regulate the replication of the subgenomic HCV replicon in Huh-7 cells. In this article, we demonstrate that Huh-7 cells harboring replicating, full-length HCV RNAs express elevated levels of ATP citrate lyase and acetyl-CoA synthetase genes, both of which are involved in cholesterol and fatty acid biosynthesis. Further, we confirm that the cholesterol-biosynthetic pathway controls HCV RNA replication by regulating the cellular levels of geranylgeranyl pyrophosphate, we demonstrate that the impact of geranylgeranylation depends on the fatty acid content of the cell, and we show that fatty acids can either stimulate or inhibit HCV replication, depending on their degree of saturation. These results illustrate a complex cellular-regulatory network that controls HCV RNA replication, presumably by modulating the trafficking and association of cellular and͞or viral proteins with cellular membranes, suggesting that pharmacologic manipulation of these pathways may have a therapeutic effect in chronic HCV infection.cholesterol ͉ replicon H epatitis C virus (HCV), a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis and hepatocellular carcinoma (1, 2). The HCV genome is a positivestranded Ϸ9.6-kb RNA molecule consisting of a single ORF, which is flanked by 5Ј and 3Ј untranslated regions (UTR). The HCV 5Ј UTR contains a highly structured internal ribosome entry site (3-8), and the 3Ј UTR is essential for replication (9, 10). The HCV ORF encodes a single 3,008-to 3,037-aa polyprotein that is posttranslationally processed to produce Ն10 different proteins: core protein, envelope proteins E1 and E2, p7, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (1,8,11). Our understanding of the biology of HCV RNA replication has been greatly facilitated by the development of subgenomic and full-length HCV replicons that express HCV proteins and replicate their RNA in stably transfected human hepatoma-cell-derived Huh-7 cells.Our laboratory has previously demonstrated (12) that multiple cellular genes involved in lipid metabolism are differentially regulated during viral spread in acutely infected chimpanzees, and that ATP citrate lyase, which is required for cholesterol and fatty acid biosynthesis, is induced during the initial rise of high-level HCV replication during acute infection in chimpanzees. There is considerable evidence suggesting that the cholesterol and fatty-acid-biosynthesis pathways may play a role in HCV replication and infection. Steatosis, i.e., the formation of hepatocellular lipid droplets, is a well documented histological characteristic of HCV infection in humans, chimpanzees, and mouse m...

Hepatitis B virus (HBV) infection is a leading risk factor for hepatocellular carcinoma (HCC). HBV integration into the host genome has been reported, but its scale, impact and contribution to HCC development is not clear. Here, we sequenced the tumor and nontumor genomes (>803 coverage) and transcriptomes of four HCC patients and identified 255 HBV integration sites. Increased sequencing to 2403 coverage revealed a proportionally higher number of integration sites. Clonal expansion of HBV-integrated hepatocytes was found specifically in tumor samples. We observe a diverse collection of genomic perturbations near viral integration sites, including direct gene disruption, viral promoterdriven human transcription, viral-human transcript fusion, and DNA copy number alteration. Thus, we report the most comprehensive characterization of HBV integration in hepatocellular carcinoma patients. Such widespread random viral integration will likely increase carcinogenic opportunities in HBV-infected individuals.[Supplemental material is available for this article.] . HBV integration into the host genome has been reported both in tumors (Gozuacik et al. 2001;Murakami et al. 2005;Saigo et al. 2008) and in nontumor liver tissue from HBV-infected individuals (Mason et al. 2010), although such integration is not essential for HBV replication. The relative extent, mutation model, and the functional impact of HBV integration in host genomes is not clear due to the lack of an unbiased approach to identify and quantify genome-wide HBV integration sites. Recent advances in sequencing technologies (Meyerson et al. 2010) provide an opportunity to investigate the global extent, mutation model, and functional impact of viral integration in the host genome. Recently, a primary hepatitis C virus-infected HCC patient has been subjected to whole-genome sequencing, and many somatic mutations were reported (Totoki et al. 2011). However, as an RNA virus, HCV never integrates into the host genome during its life cycle; therefore, liver cancer with HCV infection is not an optimal model to study viral-human genomic interactions. To that end, sequencing the genome and transcriptome of an HBV-positive HCC patient provides a great opportunity to reveal the functional impact of viral integration on the host genome.

The recent development of a cell culture infection model for hepatitis C virus (HCV) permits the production of infectious particles in vitro. In this report, we demonstrate that infectious particles are present both within the infected cells and in the supernatant. Kinetic analysis indicates that intracellular particles constitute precursors of the secreted infectious virus. Ultracentrifugation analyses indicate that intracellular infectious viral particles are similar in size (ϳ65 to 70 nm) but different in buoyant density (ϳ1.15 to 1.20 g/ml) from extracellular particles (ϳ1.03 to 1.16 g/ml). These results indicate that infectious HCV particles are assembled intracellularly and that their biochemical composition is altered during viral egress.Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Approximately 3% of the human population is infected, and more than 80% of all HCV infections progress to chronicity, ultimately leading to fibrosis, cirrhosis, and hepatocellular carcinoma (24). There is no vaccine against HCV, and the most widely used therapy involves the administration of type I interferon (␣2A) combined with ribavirin. However, this treatment strategy is toxic and has been shown to be ineffective in a significant proportion of the cases (41).HCV is a member of the Flaviviridae family and the sole member of the genus Hepacivirus (34). HCV is an enveloped virus with a single-strand positive RNA genome that codes for a unique polyprotein of approximatively 3,000 amino acids (11,12). A single open reading frame is flanked by 5Ј and 3Ј untranslated regions that contain RNA sequences essential for RNA translation and replication, respectively (17,18,23). The translation of the single open reading frame is driven by an internal ribosomal entry site sequence present within the 5Ј untranslated region (23), and the resulting polyprotein is processed by cellular and viral proteases into its individual components (reviewed in reference 42). The E1, E2, and core structural proteins are assembled into particles (3, 4), but are not essential for viral RNA replication or translation. The NS2, NS3, NS4A, NS4B, NS5A, and NS5B nonstructural proteins constitute the viral components necessary for efficient viral RNA replication, although NS2 is dispensable for this function (5, 33). In the linear sequence of the polyprotein, the structural proteins are separated from the nonstructural proteins by a small hydrophobic protein, p7 (29), whose function remains unknown but that has the potential to form ion channels (19). Viral proteins are localized in the cytoplasm, and it is assumed, by analogy with other members of the Flaviviridae family, that the entire life cycle of the virus is exclusively cytoplasmic (32).

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFN␣ in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.H epatitis C virus (HCV), a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis and hepatocellular carcinoma (1, 2). Viral clearance during acute HCV infection is usually associated with a multispecific CD4 ϩ and CD8 ϩ T cell response, which is weak or undetectable in subjects who do not control the infection (3-5). Importantly, most chronically infected patients fail to resolve HCV infection after combination therapy with .The HCV genome is a positive-stranded Ϸ9.6-kb RNA molecule consisting of a single ORF, which is flanked by 5Ј and 3Ј UTR. The HCV 5Ј UTR contains a highly structured internal ribosome entry site (8-13). The HCV ORF encodes a single polyprotein that is 3,008-3,037 aa in length and is posttranslationally modified to produce at least ten different proteins: core, envelope proteins E1 and E2, p7, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (1,13,14). Despite considerable advances in the understanding of the function of these proteins, the basic mechanism(s) of HCV replication still remain unclear because of the absence of a tissue culture system that can sustain productive virus infection (1). The recent development of subgenomic and full-length HCV replicons that replicate and express HCV proteins in stably transfected human hepatoma cell-derived Huh-7 cells has facilitated the analysis of the role of cellular pathways required in HCV replication and the efficacy of antiviral drugs (15-19). For example, by using HCV replicons, the antiviral effects of IFN␣ and IFN␥ have been clearly demonstrated (20-23). Although IFN treatment can efficiently inhibit HCV replication in cultured Huh-7 cells, Ͼ60% of patients treated with IFN do not eliminate the virus (24)(25)(26)(27)(28). This suggests that HCV may be able to induce a state of IFN resistance in the infected liver. In keeping with this notion, HCV E2 and NS5A proteins have been demonstrated to interfere with the IFN-induced signaling pathway by interacting with protein kinase R (PKR) and inhibiting its kinase activity (29-32). Thus, alternative approaches to the treatment of chronic HCV infection seem to be warranted. Double-s...

In the past several years, a number of cellular proteins have been identified as candidate entry receptors for hepatitis C virus (HCV) by using surrogate models of HCV infection. Among these, the tetraspanin CD81 and scavenger receptor B type I (SR-BI), both of which localize to specialized plasma membrane domains enriched in cholesterol, have been suggested to be key players in HCV entry. In the current study, we used a recently developed in vitro HCV infection system to demonstrate that both CD81 and SR-BI are required for authentic HCV infection in vitro, that they function cooperatively to initiate HCV infection, and that CD81-mediated HCV entry is, in part, dependent on membrane cholesterol.Hepatitis C virus (HCV) is a member of the Flaviviridae family of viruses and is a major cause of chronic hepatitis and hepatocellular carcinoma (3,15). The HCV genome is a positive-strand ϳ9.6-kb RNA molecule consisting of a single open reading frame which is flanked by 5Ј and 3Ј untranslated regions (UTR). The HCV 5Ј UTR contains a highly structured internal ribosome entry site (14,50,64,78,79,86), while the 3Ј UTR is essential for replication (32,88). The HCV open reading frame encodes a single polyprotein of 3,008 to 3,037 amino acids in length that is posttranslationally processed by host and viral proteases to produce at least 10 different proteins: core protein, envelope proteins E1 and E2, p7, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (6,15,64).The host-virus interactions required during the initial steps of HCV infection are not completely understood. Prior to the development of an in vitro infectious HCV system, HCV pseudotyped retroviral particles (HCVpp), which comprise a reporter retrovirus enclosed in an envelope containing HCV glycoproteins E1 and E2, have allowed detailed analyses of the host-virus interactions required for HCVpp entry (9,10,16,30,31,34,39,46,81,83). Using a combination of HCVpp and recombinant soluble E2 glycoprotein (sE2) binding assays, a number of cellular proteins have been identified as potential candidates for HCV entry receptors, including the tetraspanin protein CD81 (61), scavenger receptor B type I (SR-BI) (68), which is a cellular protein that binds high-density lipoprotein (HDL), the low-density lipoprotein receptor (LDL-R) (1, 52), the C-type lectins L-SIGN and DC-SIGN (25,33,48,62), heparin sulfate (8), and the asialoglycoprotein receptor (67).The roles of CD81 and SR-BI as HCVpp receptors are well documented (2, 7, 9, 11, 39, 68, 91), and CD81 was recently shown to be required for cell culture-derived HCV infection (47,85,89,92). However, the extent to which SR-BI is required for HCV infection and whether it functions cooperatively with CD81 are poorly understood.Cellular cholesterol is critical for infection by many viruses (23,26,40,53,73,87). Indeed, components of the receptor complex for dengue virus, another flavivirus, are reported to be localized to cholesterol-enriched microdomains called lipid rafts, such that depletion of cholesterol by meth...

Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody-peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the β-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies.

While epidermal growth factor receptor (EGFR) has been shown to be important in the entry process for multiple viruses, including hepatitis C virus (HCV), the molecular mechanisms by which EGFR facilitates HCV entry are not well understood. Using the infectious cell culture HCV model (HCVcc), we demonstrate that the binding of HCVcc particles to human hepatocyte cells induces EGFR activation that is dependent on interactions between HCV and CD81 but not claudin 1. EGFR activation can also be induced by antibody mediated cross-linking of CD81. In addition, EGFR ligands that enhance the kinetics of HCV entry induce EGFR internalization and colocalization with CD81. While EGFR kinase inhibitors inhibit HCV infection primarily by preventing EGFR endocytosis, antibodies that block EGFR ligand binding or inhibitors of EGFR downstream signaling have no effect on HCV entry. These data demonstrate that EGFR internalization is critical for HCV entry and identify a hitherto-unknown association between CD81 and EGFR. Hepatitis C virus (HCV), a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis and hepatocellular carcinoma (HCC) (2). While the generation of the HCV pseudoparticle (HCVpp) and infectious cell culture (HCVcc) models have resulted in a significant increase in our understanding of HCV entry, the molecular mechanisms involved in viral internalization and fusion still remain unclear. HCV entry occurs through the coordinated interactions between the E1-E2 HCV glycoproteins and at least four essential cellular entry factors: CD81 (42), scavenger receptor B type I (SR-BI) (47), occludin (OCLN) (43), and claudin 1 (CLDN1) (11). The E2 glycoprotein has been demonstrated to bind CD81 (42) and SR-BI (47), and antibodies that bind to highly conserved residues 412 to 423 on the E2 glycoprotein possess broad neutralization capabilities against multiple HCV genotypes by inhibiting HCV-CD81 interactions (40). Although HCV is known to enter hepatocytes via clathrinmediated endocytosis (1), the host-virus interactions that govern HCV internalization are not well understood. Only one of the HCV entry factors, SR-BI, enhances HCV entry by mediating the selective uptake of cholesterol esters from HDL (8). Although HCV was recently demonstrated to induce CD81 and CLDN1 endocytosis (14), the molecular interactions important for HCV internalization still remain unclear.Multiple RNA and DNA viruses have evolved to induce a variety of receptor-mediated signaling events that are critical for different aspects of viral entry (7,12,16,53). HCV regulates multiple intracellular signaling pathways, some of which been implicated in the progression of HCV-related HCC (23, 53). HCV interaction with CD81 has been demonstrated to activate multiple downstream signaling pathways, including Rho GTPase family members, Cdc42, mitogen-activated protein kinase pathways, and members of the ezrin-radixin-moesin (ERM) family of proteins (3, 6, 13). In addition, CD81 binding by HCV primes the E1-E2 heterodimer complex for low...