, indicating that CyPA is the chief mediator of the observed CsA resistance. The dependency on CyPA for replication was observed for both genotype (GT) 1a and 1b replicons as well as a GT 2a infectious virus. An interaction between CyPA and HCV RNA as well as the viral polymerase that is sensitive to CsA treatment in wild-type but not in resistant replicons was detected. These findings reveal the molecular mechanism of CsA resistance and identify CyPA as a critical cellular cofactor for HCV replication and infection.
Treatment of hepatitis C virus (HCV) infection has been met with less than satisfactory results due primarily to its resistance to and significant side effects from alpha interferon (IFN-␣). New classes of safe and broadly acting treatments are urgently needed. Cyclosporine (CsA), an immunosuppressive and anti-inflammatory drug for organ transplant patients, has recently been shown to be highly effective in suppressing HCV replication through a mechanism that is distinct from the IFN pathway. Here we report the selection and characterization of HCV replicon cells that are resistant to CsA treatment in vitro, taking advantage of our ability to sort live cells that are actively replicating HCV RNA in the presence of drug treatments. This resistance is specific to CsA as the replicon cells most resistant to CsA were still sensitive to IFN-␣ and a polymerase inhibitor. We demonstrate that the resistant phenotype is not a result of general enhanced replication and, furthermore, that mutations in the coding region of HCV NS5B contribute to the resistance. Interestingly, a point mutation (I432V) isolated from the most resistant replicon was able to rescue a lethal mutation (P540A) in NS5B that disrupts its interaction with its cofactor, cyclophilin B (CypB), even though the I432V mutation is located outside of the reported CypB binding site (amino acids 520 to 591). Our results demonstrate that CsA exerts selective pressure on the HCV genome, leading to the emergence of resistanceconferring mutations in the viral genome despite acting upon a cellular protein.Hepatitis C virus (HCV) infects more than 170 million people worldwide, leading to both acute and chronic liver diseases in patients. So far there is no prophylactic vaccine to prevent HCV infection. The current treatment, alpha interferon (IFN-␣) in combination with ribavirin (RBV), is not satisfactory because of significant side effects and resistance. Even though the mechanism of this IFN resistance is not yet completely understood, both the virus and the host probably play important roles (10, 32, 33). On one hand, specific genetic backgrounds and/or the physiological statuses of nonresponding patients may account for the failure to achieve sustained virological responses. Factors such as race, gender, age, and obesity status have all been shown to modulate the outcome of IFN therapy (10). On the other hand, the greater likelihood of developing IFN resistance by patients infected with the predominant strain of the virus, genotype 1, indicates that viral factors also influence the success rate of therapy.HCV populations in vivo exist in a quasispecies nature, likely due to the low fidelity of the RNA-dependent RNA polymerase and high turnover rate of the viral RNA. Not surprisingly, the genetic diversity of HCV in the in vitro model system, the replicon, is also very high. The majority of the HCV replicons contain cell culture-adapted mutations, and the mutation rate of long-term replicons has been estimated to be approximately 3.0 ϫ 10 Ϫ3 base substitutions/sit...
An intimate relationship between hepatitis C virus (HCV) replication and the physiological state of the host liver cells has been reported. In particular, a highly reproducible and reversible inhibitory effect of high cell density on HCV replication was observed: high levels of HCV RNA and protein can be detected in actively growing cells but decline sharply when the replicon cells reach confluence. Arrested cell growth of confluent cells has been proposed to be responsible for the inhibitory effect. Indeed, other means of arresting cell growth have also been shown to inhibit HCV replication. Here, we report a detailed study of the effect of cell growth and confluence on HCV replication using a flow cytometry-based assay that is not biased against cytostasis and reduced cell number. Although we readily reproduced the inhibitory effect of cell confluence on HCV replication, we found no evidence of inhibition by serum starvation, which arrested cell growth as expected. In addition, we observed no inhibitory effect by agents that perturb the cell cycle. Instead, our results suggest that the reduced intracellular pools of nucleosides account for the suppression of HCV expression in confluent cells, possibly through the shutoff of the de novo nucleoside biosynthetic pathway when cells become confluent. Adding exogenous uridine and cytidine to the culture medium restored HCV replication and expression in confluent cells. These results suggest that cell growth arrest is not sufficient for HCV replicon inhibition and reveal a mechanism for HCV RNA inhibition by cell confluence.Hepatitis C virus (HCV) is a positive-strand RNA virus that infects more than 170 million people worldwide. Its infection mainly affects the liver and leads to both acute and chronic liver diseases including cirrhosis and hepatocellular carcinoma. HCV replication has proved to be a good model system for studies of virus-host cell interactions, as the virus often establishes a chronic infection that typifies the intricate relationship between a pathogen and its host.Research on HCV infection in vitro has been hampered by lack of an efficient cell culture-based production and infection system. The very recent reports of production of infectious HCV particles in tissue culture will hopefully break through this barrier and usher in a new era of HCV research (15,26,32). Studies of HCV RNA replication, however, received a great boost with the development of the HCV subgenomic replicon system a number of years ago (1, 16). Many important questions about the RNA replication of the virus were answered through use of the replicon cells. Adaptive mutations that permit efficient RNA replication in cultured hepatoma cells as well as nonhepatic cell lines have been defined (1, 13, 33); critical components of replication have been mapped and the data greatly complemented the limited in vivo data available previously (5, 12, 31). A novel mechanism by which HCV suppresses the innate antiviral responses to establish persistent replication has been uncovered (4). Th...
Hepatitis C virus (HCV) infection leads to acute and chronic liver diseases, and new classes of anti-HCV therapeutics are needed. Cyclosporine A (CsA) inhibits HCV replication and CsA derivatives that lack the immunosuppressive function are currently in clinical trials as candidate anti-HCV drugs. Here we characterize several independently derived HCV replicons with varying levels of CsA resistance due to mutations in nonstructural protein 5B (NS5B), the HCV-encoded polymerase. Mutant HCV replicons engineered with these mutations showed resistance to CsA. The mutations reside in two distinct patches in the polymerase: the template channel and one face of a concave surface behind the template channel. Mutant NS5B made by cells expressing the HCV replicon had increased ability to bind to RNA in the presence of CsA. Purified recombinant NS5B proteins containing the mutations were better at de novo initiated RNA synthesis than the wild-type control. Furthermore, the mutant proteins were able to bind RNA with Ϸ8-fold higher affinity. Last, mutation near the template channel alleviated the lethal phenotype of a mutation in the concave patch, P540A. This intramolecular compensation for the HCV replicase function by amino acid changes in different domains was further confirmed in an infectious cell culture-derived virus system. C yclosporine A (CsA), a commonly used immunosuppressant for transplant patients, has recently emerged as a potential new anti-hepatitis C virus (HCV) therapeutic. CsA and its derivatives potently inhibit HCV replication both in cell-culture systems and in mice with transplanted human liver, 1-3 although no consensus has emerged on the in vivo benefits of using CsA over Tacrolimus (FK506), a compound that lacks anti-HCV effect in vitro, for HCV-infected liver-transplant patients. [4][5][6][7][8][9] More recent clinical trials with a CsA derivative, DEBIO-025, yield promising results in human immunodeficiency virus (HIV) and HCV-coinfected patients, but drug resistance in vivo has not been studied. 10 Studies of the antiviral effect of CsA on HCV replicons has led to the identification of cyclophilin (CyP) as an essential cofactor for HCV replication [11][12][13] ; point mutations in nonstructural protein 5B (NS5B) and NS5A are associated with CsA resistance in vitro. 14,15 In addition, NS5B interacts with both CyPA and CyPB both in vitro and in vivo. 12,13 These data establish NS5B as an indirect viral target for the CsA-mediated inhibition of HCV replication. The CyPA-NS5B interaction has also been shown to be the principal mediator of cyclosporine resistance in vitro. 12
Because HCV RNA-dependent RNA polymerase is error-prone and the viral RNA has a high turnover rate, the genetic diversity of HCV is very high both in vitro and in vivo. The mutation rate in long-term replicon cultures approaches 3.0 x 10(-3) base substitutions/site/year in this in vitro replication model. A direct consequence of the high mutation rate is the rapid emergence of drug-resistant variants, both in cell culture and in patients. Selectable replicons have been used extensively to isolate and characterize drug-resistant HCV genomes in vitro. Typically, replicon cells are plated at a low density and then subjected to a double selection by G418 and escalating dosages of a compound of choice. Here we describe an alternative screening assay that takes advantage of an HCV replicon that is amenable to live-cell sorting with a suitable flow cytometer. We also present a strategy for determining the relative contribution to the resistance by viral genome and host cells. We use selection and characterization of Cyclosporine A (CsA)-resistant replicons as a example to present the protocols, but this method can easily be adapted for the selection of replicon cells resistant to other chemical compounds as long as the compound does not fluoresce at the same wavelength as the fluorescent reporter protein in the replicon.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.