Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.

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...

The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.

No abstract

1054-1061.)A utophagy is important for removing long-lived proteins and damaged organelles in cells. During autophagy, double-membrane vesicles form to sequester part of the cytoplasm. These double-membrane vesicles, also known as autophagosomes, subsequently fuse with lysosomes to form autolysosomes for the degradation of their contents for recycling. 1 Many genes that are important for autophagy have been identified. Among them is microtubule-associated protein light chain 3 (LC3), whose covalent linkage to phosphatidylethanolamine by the ubiquitin-activating enzyme E1-like protein Atg7 is necessary for the formation of autophagosomes. 2 Hepatitis C virus (HCV) is a positive-stranded RNA virus with a genome size of 9.6 Kb. Infection by this virus can lead to liver cirrhosis and hepatocellular carcinoma. Based on their genetic relatedness, different HCV isolates have been grouped into six major genotypes and many more subtypes. The HCV genome codes for a polyprotein, which is proteolytically cleaved to generate the mature protein products. 3 Recently, a cell culture system for efficient HCV propagation using the JFH1 strain, which belongs to HCV genotype 2a, has been developed. [4][5][6][7] In this system, the HCV JFH1 RNA or its derivative was transfected into human hepatoma cells to direct the replication and release of infectious HCV particles, which could then initiate the next round of infection. In this report, we use this HCV RNA transfection/ infection system to study HCV-host interactions. Our results indicate that HCV induces the accumulation of autophagosomes by activating unfolded protein response (UPR). However, HCV does not enhance autophagic protein degradation. Importantly, this induction of autophagosomes enhanced HCV replication. The persistent induction of the UPR and the Abbreviations: BAF, bafilomycin A1; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; ER, endoplasmic reticulum; GFP, green fluorescence protein; HCV, hepatitis C virus; LC3, mRNA, messenger RNA; PERK, siRNA, small interfering RNA; Tg, thapsigargin; UPR, unfolded protein response. From the

Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.Hepatitis C virus (HCV) infects about 170 million people around the world. Despite the importance of HCV as a human pathogen, little is known about its cell biology. The virus was identified and cloned more than 15 years ago (7), but the lack of a robust system allowing for the production of HCV in cell culture has hampered for many years functional studies on HCV infection.In recent years, two major advances have made it possible to investigate HCV entry. A first advance has been the production of infectious retroviral particles pseudotyped with HCV envelope glycoproteins (3,14,24). Using this system of HCV pseudoparticles (HCVpp), observations on receptor usage and the facilitating role of high-density lipoprotein during entry were reported (3, 24, 55). A second major advance has been the recent development of a cell culture model for HCV (30,56,59). This system allows for the production of virus that can be efficiently propagated in cell culture (HCVcc). Therefore, the cell entry of HCV can now be investigated in the context of an infectious cycle.HCV belongs to the Hepacivirus genus in the Flaviviridae family, which also includes the Flavivirus and Pestivirus genera (31). The HCV genome encodes three structural proteins, capsid protein C and envelope glycoproteins E1 and E2, which are associated in the form of a heterodimer (13). Several cellular proteins were reported to interact in vitro with isolated E2. These putative receptors include the tetraspanin CD81 (42), the scavenger receptor class B type I (SR-BI) (50), the lectins L-SIGN and DC-SIGN (18, 32), the asialoglycoprotein receptor (49), and heparan-sulfate proteoglycans (2). The low-density lipoprotein receptor was also proposed as a candidate receptor (1). The importance of CD81 and SR-BI in HCV entry was confirmed with HCVpp (3, 4, 24) as well as with HCVcc for CD81 (30,56). Beyond receptor bind...

Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433) impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interaction and virion production, suggesting that phosphorylation of these serine residues is important for virion production. In addition, we found that the alanine substitutions in the serine cluster suppressed the association of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly. These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core interaction.Hepatitis C virus (HCV) infection is a major public health problem and is prevalent in about 200 million people worldwide (27,40,42). Current protocols for treating HCV infection fail to produce a sustained virological response in as many as half of treated individuals, and many cases progress to chronic liver disease, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (15,31,35,43).HCV is a positive-strand RNA virus classified in the Hepacivirus genus within the Flaviviridae family (55). Its approximately 9.6-kb genome is translated into a single polypeptide of about 3,000 amino acids (aa), in which the structural proteins core, E1, and E2 reside in the N-terminal region. A crucial function of core protein is assembly of the viral nucleocapsid. The amino acid sequence of this protein is well conserved among different HCV strains compared to other HCV proteins. The nonstructural (NS) proteins NS3-NS5B are considered to assemble into a membrane-associated HCV RNA replicase complex. NS3 possesses the enzymatic activities of serine protease and RNA helicase, and NS4A serves as a cofactor for NS3 protease. NS4B plays a role in the remodeling of host cell membranes, probably to generate the site for the replicase assembly. NS5B functions as the RNA-dependent RNA polymerase. NS5A is known to play an important but undefined role in viral RNA replication.NS5A is a phosphoprotein that can be...

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease (HFMD), a common febrile disease occurring mainly in young children. Although clinical manifestations of HFMD are usually mild and self limiting, a severe EV71 outbreak can lead to a diverse array of neurological diseases. Identification of the specific cellular receptors is crucial for elucidating the mechanism of early virus-host interactions and the pathogenesis of enteroviruses. Here we identify human P-selectin glycoprotein ligand-1 (PSGL-1; CD162), a sialomucin membrane protein expressed on leukocytes that has a major role in early stages of inflammation, as a functional receptor for EV71 using an expression cloning method by panning. The N-terminal region of PSGL-1 binds specifically to EV71. Stable PSGL-1 expression allowed EV71 entry and replication, and development of cytopathic effects in nonsusceptible mouse L929 cells. Five out of eight EV71 strains bound soluble PSGL-1 and used intact PSGL-1 as the primary receptor for infection of Jurkat T cells. Three other EV71 strains did not use PSGL-1, suggesting the presence of strain-specific replication of EV71 in leukocytes. EV71 replicated in nonleukocyte cell lines in a PSGL-1-independent manner, indicating the presence of alternative receptor(s) for EV71. The identification of PSGL-1 as a receptor for EV71 sheds new light on a role for PSGL-1-positive leukocytes in cell tropism and pathogenesis during the course of HFMD and other EV71-mediated diseases.