Stephanie Kallis

Characterization of the Early Steps of Hepatitis C Virus Infection by Using Luciferase Reporter Viruses

ABSTRACT The lack of an efficient system to produce hepatitis C virus (HCV) particles has impeded the analysis of the HCV life cycle. Recently, we along with others demonstrated that transfection of Huh7 hepatoma cells with a novel HCV isolate (JFH1) yields infectious viruses. To facilitate studies of HCV replication, we generated JFH1-based bicistronic luciferase reporter virus genomes. We found that RNA replication of the reporter construct was only slightly attenuated and that virus titers produced were only three- to fivefold lower compared to the parental virus, making these reporter viruses an ideal tool for quantitative analyses of HCV infections. To expand the scope of the system, we created two chimeric JFH1 luciferase reporter viruses with structural proteins from the Con1 (genotype 1b) and J6CF (genotype 2a) strains. Using these and the authentic JFH1 reporter viruses, we analyzed the early steps of the HCV life cycle. Our data show that the mode of virus entry is conserved between these isolates and involves CD81 as a key receptor for pH-dependent virus entry. Competition studies and time course experiments suggest that interactions of HCV with cell surface-resident glycosaminoglycans aid in efficient infection of Huh7 cells and that CD81 acts during a postattachment step. The reporter viruses described here should be instrumental for investigating the viral life cycle and for the development of HCV inhibitors.

Essential Role of Domain III of Nonstructural Protein 5A for Hepatitis C Virus Infectious Particle Assembly

Persistent infection with the hepatitis C virus (HCV) is a major risk factor for the development of liver cirrhosis and hepatocellular carcinoma. With an estimated about 3% of the world population infected with this virus, the lack of a prophylactic vaccine and a selective therapy, chronic hepatitis C currently is a main indication for liver transplantation. The establishment of cell-based replication and virus production systems has led to first insights into the functions of HCV proteins. However, the role of nonstructural protein 5A (NS5A) in the viral replication cycle is so far not known. NS5A is a membrane-associated RNA-binding protein assumed to be involved in HCV RNA replication. Its numerous interactions with the host cell suggest that NS5A is also an important determinant for pathogenesis and persistence. In this study we show that NS5A is a key factor for the assembly of infectious HCV particles. We specifically identify the C-terminal domain III as the primary determinant in NS5A for particle formation. We show that both core and NS5A colocalize on the surface of lipid droplets, a proposed site for HCV particle assembly. Deletions in domain III of NS5A disrupting this colocalization abrogate infectious particle formation and lead to an enhanced accumulation of core protein on the surface of lipid droplets. Finally, we show that mutations in NS5A causing an assembly defect can be rescued by trans-complementation. These data provide novel insights into the production of infectious HCV and identify NS5A as a major determinant for HCV assembly. Since domain III of NS5A is one of the most variable regions in the HCV genome, the results suggest that viral isolates may differ in their level of virion production and thus in their level of fitness and pathogenesis.

Hepatitis C Virus p7 Protein Is Crucial for Assembly and Release of Infectious Virions

Hepatitis C virus (HCV) infection is associated with chronic liver disease and currently affects about 3% of the world population. Although much has been learned about the function of individual viral proteins, the role of the HCV p7 protein in virus replication is not known. Recent data, however, suggest that it forms ion channels that may be targeted by antiviral compounds. Moreover, this protein was shown to be essential for infectivity in chimpanzee. Employing the novel HCV infection system and using a genetic approach to investigate the function of p7 in the viral replication cycle, we find that this protein is essential for efficient assembly and release of infectious virions across divergent virus strains. We show that p7 promotes virus particle production in a genotype-specific manner most likely due to interactions with other viral factors. Virus entry, on the other hand, is largely independent of p7, as the specific infectivity of released virions with a defect in p7 was not affected. Together, these observations indicate that p7 is primarily involved in the late phase of the HCV replication cycle. Finally, we note that p7 variants from different isolates deviate substantially in their capacity to promote virus production, suggesting that p7 is an important virulence factor that may modulate fitness and in turn virus persistence and pathogenesis.

Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Hepatitis C Virus Replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.

Essential Role of Cyclophilin A for Hepatitis C Virus Replication and Virus Production and Possible Link to Polyprotein Cleavage Kinetics

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

The Level of CD81 Cell Surface Expression Is a Key Determinant for Productive Entry of Hepatitis C Virus into Host Cells

ABSTRACT Recently a cell culture model supporting the complete life cycle of the hepatitis C virus (HCV) was developed. Searching for host cell determinants involved in the HCV replication cycle, we evaluated the efficiency of virus propagation in different Huh-7-derived cell clones. We found that Huh-7.5 cells and Huh7-Lunet cells, two former replicon cell clones that had been generated by removal of an HCV replicon by inhibitor treatment, supported comparable levels of RNA replication and particle production, whereas virus spread was severely impaired in the latter cells. Analysis of cell surface expression of CD81 and scavenger receptor class B type I (SR-BI), two molecules previously implicated in HCV entry, revealed similar expression levels for SR-BI, while CD81 surface expression was much higher on Huh-7.5 cells than on Huh7-Lunet cells. Ectopic expression of CD81 in Huh7-Lunet cells conferred permissiveness for HCV infection to a level comparable to that for Huh-7.5 cells. Modulation of CD81 cell surface density in Huh-7.5 cells by RNA interference indicated that a certain amount of this molecule (∼7 × 104 molecules per cell) is required for productive infection with a low dose of HCV. Consistent with this, we show that susceptibility to HCV infection depends on a critical quantity of CD81 molecules. While infection is restricted in cells expressing very small amounts of CD81, susceptibility rapidly rises within a narrow range of CD81 levels, reaching a plateau where higher expression does not further increase the efficiency of infection. Together these data indicate that a high density of cell surface-exposed CD81 is a key determinant for productive HCV entry into host cells.

Analysis of Hepatitis C Virus Superinfection Exclusion by Using Novel Fluorochrome Gene-Tagged Viral Genomes

ABSTRACT Studies of the complete hepatitis C virus (HCV) life cycle have become possible with the development of an infectious cell culture system using the genotype 2a isolate JFH-1. Taking advantage of this system in the present study, we investigated whether HCV infection leads to superinfection exclusion, a state in which HCV-infected cells are resistant to secondary HCV infection. To discriminate between viral genomes, we inserted genes encoding fluorescent proteins in frame into the 3′-terminal NS5A coding region. These genomes replicated to wild-type levels and supported the production of infectious virus particles. Upon simultaneous infection of Huh-7 cells, coreplication of both viral genomes in the same cell was detected. However, when infections were performed sequentially, secondary infection was severely impaired. This superinfection exclusion was neither due to a reduction of cell surface expression of CD81 and scavenger receptor BI, two molecules implicated in HCV entry, nor due to a functional block at the level of virus entry. Instead, superinfection exclusion was mediated primarily by interference at the level of HCV RNA translation and, presumably, also replication. In summary, our results describe the construction and characterization of viable monocistronic HCV reporter genomes allowing detection of viral replication in infected living cells. By using these genomes, we found that HCV induces superinfection exclusion, which is primarily due to interference at a postentry step.

Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations.

With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.

Efficient trans-Encapsidation of Hepatitis C Virus RNAs into Infectious Virus-Like Particles

ABSTRACT Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteins trans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, these trans-complemented HCV particles (HCVTCP) penetrate target cells in a CD81 receptor-dependent fashion. Since HCVTCP production was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCVTCP stocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 106 tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excluding cis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCVTCP may prove valuable for gene delivery or vaccination approaches.

Antiviral effects of amantadine and iminosugar derivatives against hepatitis C virus

Current therapy of chronic hepatitis C is based on the combination of pegylated interferon‐α and ribavirin. In spite of 50% sustained virological response, therapy is still limited by unsatisfying success rates with genotype 1 infections and adverse side effects. One attempt to increase success rates is triple combination therapy of interferon and ribavirin with amantadine, a drug assumed to interfere with HCV p7 ion channel function. However, results from clinical trials indicate limited efficacy and the antiviral activity is unclear. In contrast, NS3 protease inhibitors have shown potent antiviral effects in clinical trials but rapid selection for drug resistance may limit their benefit. Targeting cellular factors required for HCV is therefore an attractive alternative. In this study, employing a system for production of infectious HCV particles in cell culture, we determined the antiviral effects of amantadine and iminosugar derivatives; the second of which primarily target host cell glucosidases required for folding and maturation of HCV envelope glycoproteins. We found that across a spectrum of HCV isolates and genotypes, amantadine affected neither RNA replication nor the release or infectivity of HCV particles. In agreement, p7 ion channel activity was not affected by amantadine, demonstrating that amantadine is not an HCV‐selective antiviral. In contrast, a dose‐dependent reduction of virus titers was achieved with iminosugars. Furthermore, HCV was rapidly eliminated from cell culture upon passage in the presence of a long alkyl chain deoxynojirimycin (DNJ). Conclusion: Iminosugar derivatives are potential drugs for treatment of HCV infections. (HEPATOLOGY 2007.)