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...

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...

Mobile group II introns can be retargeted to insert into virtually any desired DNA target. Here we show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1-22%. Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations. Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria.

Hepatitis C virus (HCV) infection is dependent on at least three coreceptors: CD81, scavenger receptor BI (SR-BI), and claudin-1. The mechanism of how these molecules coordinate HCV entry is unknown. In this study we demonstrate that a cell culture-adapted JFH-1 mutant, with an amino acid change in E2 at position 451 (G451R), has a reduced dependency on SR-BI. This altered receptor dependency is accompanied by an increased sensitivity to neutralization by soluble CD81 and enhanced binding of recombinant E2 to cell surface-expressed and soluble CD81. Fractionation of HCV by density gradient centrifugation allows the analysis of particle-lipoprotein associations. The cell culture-adapted mutation alters the relationship between particle density and infectivity, with the peak infectivity occurring at higher density than the parental virus. No association was observed between particle density and SR-BI or CD81 coreceptor dependence. JFH-1 G451R is highly sensitive to neutralization by gp-specific antibodies, suggesting increased epitope exposure at the virion surface. Finally, an association was observed between JFH-1 particle density and sensitivity to neutralizing antibodies (NAbs), suggesting that lipoprotein association reduces the sensitivity of particles to NAbs. In summary, mutation of E2 at position 451 alters the relationship between particle density and infectivity, disrupts coreceptor dependence, and increases virion sensitivity to receptor mimics and NAbs. Our data suggest that a balanced interplay between HCV particles, lipoprotein components, and viral receptors allows the evasion of host immune responses.Hepatitis C virus (HCV), the sole member of the Hepacivirus genus within the Flaviviridae, poses a global health burden, with an estimated 170 million infected individuals (according to the WHO). The majority of patients suffer a chronic infection that is associated with a progressive liver disease (1). HCV has a short positive-sense RNA genome encoding three structural (core protein, E1, and E2) glycoproteins (gps) and seven nonstructural proteins (p7 and NS2 to NS5) (40). The E1 and E2 gps interact with cell surface receptors to facilitate particle entry via low-pH and clathrin-dependent endocytosis (9,15,29,47,71). The recent discovery that the JFH-1 strain of HCV can replicate and assemble infectious particles in cultured cells (HCVcc) has allowed investigation into the viral life cycle for the first time since its identification almost 20 years ago (41,75,79).Early studies with truncated soluble HCV E2 (sE2) identified interactions with the tetraspanin CD81 and scavenger receptor class B type I (SR-BI) (56, 62). The recent availability of HCVcc and HCV pseudoparticles (HCVpp) provided the tools to validate receptor candidates. HCV entry is thought to require at least three cellular receptors: CD81, SR-BI, and the tight junction protein claudin-1 (reviewed in references 21 and 74). Other candidate components include glycosaminoglycans (5, 6, 51), low-density lipoprotein receptor (49,77), and the C...

Mobile group II introns have been used to develop a novel class of gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. Here, we have developed a targetron containing a retrotransposition-activated selectable marker (RAM), which enables one-step bacterial gene disruption at near 100% efficiency after selection. The targetron can be generated via PCR without cloning, and after intron integration, the marker gene can be excised by recombination between flanking Flp recombinase sites, enabling multiple sequential disruptions. We also show that a RAM-targetron with randomized target site recognition sequences yields single insertions throughout the Escherichia coli genome, creating a gene knockout library. Analysis of the randomly selected insertion sites provides further insight into group II intron target site recognition rules. It also suggests that a subset of retrohoming events may occur by using a primer generated during DNA replication, and reveals a previously unsuspected bias for group II intron insertion near the chromosome replication origin. This insertional bias likely reflects at least in part the higher copy number of origin proximal genes, but interaction with the replication machinery or other features of DNA structure or packaging may also contribute.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.

No abstract