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...

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.

In addition to its cellular homeostasis function, autophagy is emerging as a central component of antimicrobial host defense against diverse infections. To counteract this mechanism, many pathogens have evolved to evade, subvert, or exploit autophagy. Here, we report that autophagy proteins (i.e., Beclin-1, Atg4B, Atg5, and Atg12) are proviral factors required for translation of incoming hepatitis C virus (HCV) RNA and, thereby, for initiation of HCV replication, but they are not required once infection is established. These results illustrate a previously unappreciated role for autophagy in the establishment of a viral infection and they suggest that different host factors regulate the translation of incoming viral genome and translation of progeny HCV RNA once replication is established.H CV infection is a leading cause of chronic liver disease world-wide. With 180 million persistently infected people, chronic hepatitis C infection, which induces end stage liver diseases, such as liver cirrhosis and hepatocellular carcinoma (HCC), represents a major public health problem of high socioeconomic impact (1). However, treatment options for chronic hepatitis C are limited and a vaccine against HCV is not available. Thus, efforts to elucidate the details of the host-virus relationship during HCV infection are needed to develop efficient therapeutic strategies against this major human pathogen.HCV is an enveloped, positive strand RNA virus in the Flaviviridae family. The HCV genome is approximately 9.6 kb in length and consists of a single ORF flanked by untranslated regions (UTR) (2). Translation of the single ORF is driven by an internal ribosomal entry site (IRES) sequence present within the 5ЈUTR. The resulting polyprotein is processed by cellular and viral proteases into the structural proteins (core, E1, and E2) and the nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (2). HCV NS3 to NS5B proteins are necessary and sufficient to establish membrane-bound replication complexes that catalyze RNA replication (2).Autophagy is an intracellular degradation process in which cytoplasmic components, including organelles, are directed to the lysosome by a membrane-mediated process (3). More than 31 autophagy-related genes (ATG) whose products regulate autophagy have been identified, primarily through yeast genetics, and several have mammalian counterparts (3). They regulate distinct aspects of autophagic vesicle formation and maturation, including the assembly of an isolation membrane around a portion of cytoplasm, completion of a double-membranesurrounded autophagosome, and lysosomal fusion with degradation of its contents (3). Importantly, many studies demonstrate that autophagy is activated upon viral or bacterial infection (4). In Sindbis virus, tobacco mosaic virus, herpes simplex virus type 1 (HSV-1) and several bacterial infections, autophagy may have a protective function by restricting intracellular pathogen replication or by ensuring the survival of infected and/or uninfected cells (4). In this r...

Intracellular infectious hepatitis C virus (HCV) particles display a distinctly higher buoyant density than do secreted virus particles, suggesting that the characteristic low density of extracellular HCV particles is acquired during viral egress. We took advantage of this difference to examine the determinants of assembly, maturation, degradation, and egress of infectious HCV particles. The results demonstrate that HCV assembly and maturation occur in the endoplasmic reticulum (ER) and post-ER compartments, respectively, and that both depend on microsomal transfer protein and apolipoprotein B, in a manner that parallels the formation of very-low-density lipoproteins (VLDL). In addition, they illustrate that only low-density particles are efficiently secreted and that immature particles are actively degraded, in a proteasome-independent manner, in a post-ER compartment of the cell. These results suggest that by coopting the VLDL assembly, maturation, degradation, and secretory machinery of the cell, HCV acquires its hepatocyte tropism and, by mimicry, its tendency to persist.Hepatitis C virus (HCV) establishes persistent infection in Ͼ70% of infected individuals (25), and over 170 million people are persistently infected worldwide. Persistent HCV infection is associated with a chronic inflammatory disease (hepatitis) that ultimately leads to hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (25). Chronic infection is also associated with disorders of lipid metabolism (54), with abnormal accumulation of lipids in the liver parenchymal cells (steatosis) and reduced serum beta-lipoprotein levels (52). Currently, the only approved antiviral therapy for HCV is the administration of type I interferon combined with ribavirin. However, this therapy is toxic and is effective in only a fraction of cases (43).HCV is the sole member of the genus Hepacivirus, which belongs to the Flaviviridae family. The virus is enveloped, and the single-stranded positive-strand RNA genome contains a single open reading frame flanked by untranslated regions (5Ј UTR and 3Ј UTR) that contain RNA sequences essential for RNA translation and replication (17,18). Translation of the single open reading frame is driven by an internal ribosomal entry site (IRES) sequence present within the 5Ј UTR (24), and the resulting polyprotein, of approximately 3,000 amino acids in length, is processed by cellular and viral proteases into its individual components (44). The nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B are sufficient to support efficient HCV RNA replication in membranous compartments in the cytosol (10,35,39). Overexpression of the core, E1, and E2 proteins is sufficient for the formation of virus-like structures in insect cells (6), and expression of the viral polyprotein leads to the formation of virus-like particles in HeLa (38) and Huh-7 (23) cells. It has been proposed that infectious particles are assembled when genomic RNA-containing core particles bud through the endoplasmic reticulum (ER) membrane (47), acquiring the v...

Hepatitis C virus (HCV), a member of the Flaviviridae family, is a single-stranded positive-sense RNA virus that infects >170 million people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite its ability to block the innate host response in infected hepatocyte cell lines in vitro, HCV induces a strong type 1 interferon (IFN) response in the infected liver. The source of IFN in vivo and how it is induced are currently undefined. Here we report that HCV-infected cells trigger a robust IFN response in plasmacytoid dendritic cells (pDCs) by a mechanism that requires active viral replication, direct cell-cell contact, and Toll-like receptor 7 signaling, and we show that the activated pDC supernatant inhibits HCV infection in an IFN receptor-dependent manner. Importantly, the same events are triggered by HCV subgenomic replicon cells but not by free virus particles, suggesting the existence of a novel cell-cell RNA transfer process whereby HCV-infected cells can activate pDCs to produce IFN without infecting them. These results may explain how HCV induces IFN production in the liver, and they reveal a heretofore unsuspected aspect of the innate host response to viruses that can subvert the classical sensing machinery in the cells they infect, and do not infect or directly activate pDCs.innate immune response | TLR7 | Toll-like receptor | hepatocyte H epatitis C virus (HCV), a member of the Flaviviridae family, is a single-stranded positive-sense RNA virus that causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (1). Type 1 interferons (IFNα/β) play critical roles in the defense against virus infection. The HCV NS3/4A protease strongly inhibits type 1 IFN induction in infected cells by cleaving a key intermediate in the double-stranded RNA (dsRNA) signaling pathway (2-4). Nonetheless, HCV strongly induces IFN-stimulated gene (ISG) expression in the infected liver (5-7). The discrepancy between these observations suggests that type 1 IFNs may be produced by liver cells other than infected hepatocytes. Plasmacytoid dendritic cells (pDCs) are a highly specialized subset of dendritic cells that produce type 1 IFNs in response to microbial stimuli (8,9) and are abundant in the HCV-infected liver (10). Although HCV has been reported to suppress pDC numbers and function (11-13), their role in the control of HCV infection has not been examined. Here we show that pDCs produce large amounts of type 1 IFN via Toll-like receptor 7 (TLR7) signaling that is induced by direct cell-to-cell contact with HCV-infected cells. Importantly, these events require viral RNA replication but not virion formation in the stimulator cells. These results could explain how IFN is produced during natural HCV infection, and they reveal a host response mechanism to HCV and possibly other viruses that do not infect or directly activate pDCs.

The virological and cellular consequences of persistent hepatitis C virus (HCV) infection have been elusive due to the absence of the requisite experimental systems. Here, we report the establishment and the characteristics of persistent in vitro infection of human hepatoma-derived cells by a recently described HCV genotype 2a infectious molecular clone. Persistent in vitro infection was characterized by the selection of viral variants that displayed accelerated expansion kinetics, higher peak titers, and increased buoyant densities. Sequencing analysis revealed the selection of a single adaptive mutation in the HCV E2 envelope protein that was largely responsible for the variant phenotype. In parallel, as the virus became more aggressive, cells that were resistant to infection emerged, displaying escape mechanisms operative at the level of viral entry, HCV RNA replication, or both. Collectively, these results reveal the existence of coevolutionary events during persistent HCV infection that favor survival of both virus and host.The hepatitis C virus (HCV) is a hepatotropic, positivestranded RNA virus that causes acute and chronic hepatitis. Because most infections become persistent, HCV chronically infects more than 170 million people worldwide, many of whom will develop liver cirrhosis and hepatocellular carcinoma (15). HCV is thought to be noncytopathic in vivo, and the pathogenesis of the associated hepatitis is assumed to reflect destruction of HCV infected cells by cytotoxic CD8 ϩ T cells (9). HCV is the sole member of the genus Hepacivirus in the Flaviviridae family. Its 9.6-kb RNA genome encodes a long open reading frame that is co-and posttranslationally cleaved by cellular and viral proteases into structural (core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (2). The viral life cycle and the host-virus interactions that determine the outcome of HCV infection have been difficult to study due to the absence of a tissue culture model of HCV infection. Recently, several groups (16,20,25,29,31) have developed cell culture models of HCV infection that release HCV particles that are infectious for human hepatoma-derived cell lines. The most robust of these in vitro infections are based on the extraordinary replicative capacity of the genotype 2a JFH-1 strain of HCV, which replicates efficiently in vitro without requiring adaptive mutations (14). Importantly, cell culture-derived JFH-1 and a chimeric virus expressing the structural region of the related J6 strain of HCV and the nonstructural region of JFH-1 are infectious for chimpanzees and uPA-SCID mice reconstituted with human hepatocytes (17,25).At present, the cell culture system has been used primarily to study the early steps of HCV infection. For example, we and others (16, 31) have reported that primary HCV infection can be inhibited by blocking the interaction between the HCV E2 glycoprotein and the cellular protein CD81, an important coreceptor for HCV entry (3,13,18,30). In the current study, we used the cell c...

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).

An amphipathic ␣-helical peptide (C5A) derived from the membrane anchor domain of the hepatitis C virus (HCV) NS5A protein is virocidal for HCV at submicromolar concentrations in vitro. C5A prevents de novo HCV infection and suppresses ongoing infection by inactivating both extra-and intracellular infectious particles, and it is nontoxic in vitro and in vivo at doses at least 100-fold higher than required for antiviral activity. Mutational analysis indicates that C5A's amphipathic ␣-helical structure is necessary but not sufficient for its virocidal activity, which depends on its amino acid composition but not its primary sequence or chirality. In addition to HCV, C5A inhibits infection by selected flaviviruses, paramyxoviruses, and HIV. These results suggest a model in which C5A destabilizes viral membranes based on their lipid composition, offering a unique therapeutic approach to HCV and other viral infections.HCV ͉ amphipathic peptide ͉ antiviral peptide ͉ NS5A ͉ HIV H epatitis C virus (HCV), a member of the Flaviviridae family (1), is a single-stranded positive-sense RNA virus that causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (2, 3). HCV infects Ͼ170 million people worldwide and is the most common cause of liver transplantation in the United States (3). There is no vaccine available for HCV, and the only currently approved treatment (combination therapy with IFN and ribavirin) has limited efficacy and serious side effects (4, 5). Thus, development of new classes of antiviral compounds with improved efficacy and toxicity profiles is urgently needed.The development of HCV replicon technology several years ago (6) greatly accelerated the pace of antiviral drug discovery, leading to the development of HCV protease and polymerase inhibitors that are currently under clinical evaluation (7,8). The landscape for drug discovery improved further with the establishment of a cell culture model of HCV infection in 2005 (9-11), making it possible to search for inhibitors of every step in the HCV life cycle and agents that target the virus itself. We now report the discovery of several HCV-derived synthetic peptides that inhibit HCV infection in the cell culture infection system. One of those inhibitory peptides, an amphipathic ␣-helical 18-mer derived from the membrane anchor domain of the HCV nonstructural protein NS5A that was particularly potent against HCV and selected other virus infections, serves as the basis of this report. Results Identification of Antiviral Peptides.A peptide library of 441 overlapping peptides (18-mers offset by 11 amino acids) covering the entire HCV polyprotein (H77 strain, genotype 1a) was screened (20 M) for the ability to inhibit HCV infection (JFH-1) in a focus reduction assay using Huh-7.5.1 cells (Fig. 1). Thirteen peptides were shown to inhibit HCV focus formation by Ͼ90%. Validation of the antiviral activity of the 13 inhibitory peptides was performed by comparing the ability of each peptide (20 M) to inhibit the expansion of HCV RNA in Huh-7.5.1 cells ...