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...

RNA interference through non-coding microRNAs (miRNAs) represents a vital component of the innate antiviral immune response in plants and invertebrate animals; however, a role for cellular miRNAs in the defence against viral infection in mammalian organisms has thus far remained elusive 1 . Here we show that interferon beta (IFNβ) rapidly modulates the expression of numerous cellular miRNAs, and that eight of these IFNβ-induced miRNAs have sequence-predicted targets within the hepatitis C virus (HCV) genomic RNA. The introduction of synthetic miRNA-mimics corresponding to these IFNβ-induced miRNAs reproduces the antiviral effects of IFNβ on HCV replication and infection, whereas neutralization of these antiviral miRNAs with anti-miRNAs reduces the antiviral effects of IFNβ against HCV. In addition, we demonstrate that IFNβ treatment leads to a significant reduction in the expression of the liver-specific miR-122, an miRNA that has been previously shown to be essential for HCV replication 2 . Therefore, our findings strongly support the notion that mammalian organisms too, through the interferon system, use cellular miRNAs to combat viral infections. miRNAs are a class of small non-coding RNA molecules that function through posttranscriptional regulation of gene expression by a process termed RNA interference (RNAi). Over 500 miRNA-encoding genes, which seem to be exclusively transcribed by RNA polymerase II, have been identified in mammals. These primary microRNAs are processed by the enzymes Drosha/DGCR8/RNASEN into hairpin-loop-containing pre-miRNAs, which are then subject to nuclear export via exportin 5. Further enzymatic processing of the pre-miRNAs by Dicer leads to a mature miRNA duplex that is loaded into the RNA-induced silencing complex (RISC), in which the miRNA guides RISC to complementary messenger RNAs. On the basis of the degree of homology between the miRNA and the mRNA, RISC can inhibit

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...

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...

No abstract