Processing of GB virus B non-structural proteins in cultured cells requires both NS3 protease and NS4A cofactor

The identification of antivirals and vaccines against hepatitis C virus (HCV) infection is hampered by the lack of convenient animal models. The need to develop surrogate models has recently drawn attention to GB virus B (GBV-B), which produces hepatitis in small primates. In a previous study in vitro, it was shown that GBV-B NS3 protease shares substrate specificity with the HCV enzyme, known to be crucial for virus replication. In this report, GBV-B NS3 activity on GBV-B precursor proteins has been analysed in a cell-based system. It is shown that mature protein products are obtained that are compatible with the cleavage sites proposed on the basis of sequence homology with HCV and that GBV-B NS4A protein is required as a cofactor for optimal enzymatic activity. Experiments in vitro supported by a structural model mapped the region of NS4A that interacts with NS3 and showed that the GBV-B cofactor cannot be substituted for by its HCV analogue.

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A New In Vitro Model for Predicting the Toxicity of Cosmetic Products

Alternatives to Laboratory Animals ◽

10.1177/026119299202000119 ◽

1992 ◽

Vol 20 (1) ◽

pp. 138-143

Author(s):

Maria Carrara ◽

Lorenzo Cima ◽

Roberto Cerini ◽

Maurizio Dalle Carbonare

Keyword(s):

Cell Culture ◽

Cultured Cells ◽

Neutral Red ◽

Total Protein Content ◽

Cosmetic Products ◽

Cell Culture Insert ◽

A Cell ◽

Vitro Model ◽

Cosmetic Emulsions

A method has been developed whereby cosmetic products which are not soluble in water or in alcohol can be brought into contact with cell cultures by being placed in a cell culture insert, which is then placed in the cell culture well. Preliminary experiments were carried out with L929 cells, and cytotoxicity was evaluated by measuring neutral red uptake and the total protein content of treated cultured cells. Encouraging results were obtained in comparisons of three cosmetic emulsions and of one emulsion containing a range of concentrations of two preservatives, Kathon CG and Bronopol.

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Identification of Hepatitis C Virus NS5A Inhibitors

Journal of Virology ◽

10.1128/jvi.01360-09 ◽

2009 ◽

Vol 84 (1) ◽

pp. 482-491 ◽

Cited By ~ 137

Author(s):

Julie A. Lemm ◽

Donald O'Boyle ◽

Mengping Liu ◽

Peter T. Nower ◽

Richard Colonno ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Therapeutic Index ◽

Dna Viruses ◽

Genotype 1A ◽

A Cell ◽

Ns3 Protease ◽

Derivatives Of ◽

Rna And Dna

ABSTRACT Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was ∼5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.

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In vitro hepatitis C virus infection and hepatic choline metabolism

10.1101/746776 ◽

2019 ◽

Author(s):

Kaelan Gobeil Odai ◽

Conor O’Dwyer ◽

Rineke Steenbergen ◽

Tyler A. Shaw ◽

Tyler M. Renner ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cultured Cells ◽

Hcv Infection ◽

Choline Uptake ◽

Choline Transport ◽

Choline Metabolism ◽

Positive Strand Rna ◽

Uptake And Metabolism

AbstractCholine is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC) and can enter one carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses can impact phospholipid metabolism. In the current study we sought to interrogate the link between choline transport and early HCV infection. Namely, we aimed to investigate how HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing how the inhibition of choline uptake and metabolism upon concurrent HCV infection may alter early viral replication. Finally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter choline transporter-like family (CTL), and that CTL1 expression and the incorporation of choline into PC is diminished in 24 h infected FBS-cultured cells. Reciprocally, limiting the availability of choline for PC synthesis resulted in increased HCV replication at this early stage. In chronically HS-cultured Huh7.5 cells, there were no differences in the expression of choline transporters upon HCV infection or alterations to viral replication when choline transport was inhibited compared to control treatments. However, inhibiting choline uptake and metabolism in this system significantly impaired the production of infectious virions in HS-cultured cells. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.Abstract Figure

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Discovery of a series of novel compounds with moderate anti-hepatitis C virus NS3 protease activity in vitro

Bioorganic & Medicinal Chemistry ◽

10.1016/j.bmc.2015.07.032 ◽

2015 ◽

Vol 23 (17) ◽

pp. 5539-5545 ◽

Cited By ~ 4

Author(s):

Fangyuan Shi ◽

Yingjie Zhang ◽

Wenfang Xu

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Activity ◽

Ns3 Protease

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Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are trans-Packaged In Vitro To Generate Infectious Defective Particles

Journal of Virology ◽

10.1128/jvi.00308-09 ◽

2009 ◽

Vol 83 (18) ◽

pp. 9079-9093 ◽

Cited By ~ 32

Author(s):

Laura Pacini ◽

Rita Graziani ◽

Linda Bartholomew ◽

Raffaele De Francesco ◽

Giacomo Paonessa

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Line ◽

Genome Structure ◽

Structural Proteins ◽

Wild Type Virus ◽

Deletion Mutants ◽

Wild Type ◽

Naturally Occurring

ABSTRACT Naturally occurring hepatitis C virus (HCV) subgenomic RNAs have been found in several HCV patients. These subgenomic deletion mutants, mostly lacking the genes encoding envelope glycoproteins, were found in both liver and serum, where their relatively high abundance suggests that they are capable of autonomous replication and can be packaged and secreted in viral particles, presumably harboring the envelope proteins from wild type virus coinfecting the same cell. We recapitulated some of these natural subgenomic deletions in the context of the isolate JFH-1 and confirmed these hypotheses in vitro. In Huh-7.5 cells, these deletion-containing genomes show robust replication and can be efficiently trans-packaged and infect naïve Huh-7.5 cells when cotransfected with the full-length wild-type J6/JFH genome. The genome structure of these natural subgenomic deletion mutants was dissected, and the maintenance of both core and NS2 regions was proven to be significant for efficient replication and trans-packaging. To further explore the requirements needed to achieve trans-complementation, we provided different combinations of structural proteins in trans. Optimal trans-complementation was obtained when fragments of the polyprotein encompassing core to p7 or E1 to NS2 were expressed. Finally, we generated a stable helper cell line, constitutively expressing the structural proteins from core to p7, which efficiently supports trans-complementation of a subgenomic deletion encompassing amino acids 284 to 732. This cell line can produce and be infected by defective particles, thus representing a powerful tool to investigate the life cycle and relevance of natural HCV subgenomic deletion mutants in vivo.

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In Vitro Hepatitis C Virus Infection and Hepatic Choline Metabolism

Viruses ◽

10.3390/v12010108 ◽

2020 ◽

Vol 12 (1) ◽

pp. 108 ◽

Cited By ~ 1

Author(s):

Kaelan Gobeil Odai ◽

Conor O’Dwyer ◽

Rineke Steenbergen ◽

Tyler A. Shaw ◽

Tyler M. Renner ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Post Infection ◽

Cultured Cells ◽

Hcv Infection ◽

Choline Uptake ◽

Choline Metabolism ◽

Positive Strand Rna ◽

Uptake And Metabolism

Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC), and can enter one-carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses and can impact phospholipid metabolism. In the current study we sought to interrogate if HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing if the inhibition of choline uptake and metabolism upon concurrent HCV infection alters viral replication and infectivity. Additionally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS- and HS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter, choline transporter-like family (CTL). HCV infection in FBS, but not HS-cultured cells diminished CTL1 transcript and protein expression at 24 h post-infection, which was associated with lower choline uptake and lower incorporation of choline into PC. No changes in other transporters were observed and at 96 h post-infection, all differences were normalized. Reciprocally, limiting the availability of choline for PC synthesis by use of a choline uptake inhibitor resulted in increased HCV replication at this early stage (24 h post-infection) in both FBS- and HS-cultured cells. Finally, in chronic infection (96 h post-infection), inhibiting choline uptake and metabolism significantly impaired the production of infectious virions. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.

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Preclinical Characterization of the Novel Hepatitis C Virus NS3 Protease Inhibitor GS-9451

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00487-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 647-653 ◽

Cited By ~ 25

Author(s):

Huiling Yang ◽

Margaret Robinson ◽

Amoreena C. Corsa ◽

Betty Peng ◽

Guofeng Cheng ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Activity ◽

Protease Inhibitor ◽

Hcv Infection ◽

Cross Resistance ◽

Protease Gene ◽

Ns5a Inhibitor ◽

Ns3 Protease

ABSTRACTGS-9451 is a selective hepatitis C virus (HCV) NS3 protease inhibitor in development for the treatment of genotype 1 (GT1) HCV infection. Key preclinical properties of GS-9451, includingin vitroantiviral activity, selectivity, cross-resistance, and combination activity, as well as pharmacokinetic properties, were determined. In multiple GT1a and GT1b replicon cell lines, GS-9451 had mean 50% effective concentrations (EC50s) of 13 and 5.4 nM, respectively, with minimal cytotoxicity; similar potency was observed in chimeric replicons encoding the NS3 protease gene of GT1 clinical isolates. GS-9451 was less active in GT2a replicon cells (EC50= 316 nM). Additive to synergisticin vitroantiviral activity was observed when GS-9451 was combined with other agents, including alpha interferon, ribavirin, and the polymerase inhibitors GS-6620 and tegobuvir (GS-9190), as well as the NS5A inhibitor ledipasvir (GS-5885). GS-9451 retained wild-type activity against multiple classes of NS5B and NS5A inhibitor resistance mutations. GS-9451 was stable in hepatic microsomes and hepatocytes from human and three other tested species. Systemic clearance was low in dogs and monkeys but high in rats. GS-9451 showed good oral bioavailability in all three species tested. In rats, GS-9451 levels were ∼40-fold higher in liver than plasma after intravenous dosing, and elimination of GS-9451 was primarily through biliary excretion. Together, these results are consistent with the antiviral activity observed in a recent phase 1b study. The results ofin vitrocross-resistance and combination antiviral assays support the ongoing development of GS-9451 in combination with other agents for the treatment of chronic HCV infection.

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Differential Biophysical Properties of Infectious Intracellular and Secreted Hepatitis C Virus Particles

Journal of Virology ◽

10.1128/jvi.01150-06 ◽

2006 ◽

Vol 80 (22) ◽

pp. 11074-11081 ◽

Cited By ~ 186

Author(s):

Pablo Gastaminza ◽

Sharookh B. Kapadia ◽

Francis V. Chisari

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Buoyant Density ◽

Infection Model ◽

Virus Particles ◽

Viral Egress ◽

Viral Particles ◽

A Cell ◽

Infected Cells

ABSTRACT The recent development of a cell culture infection model for hepatitis C virus (HCV) permits the production of infectious particles in vitro. In this report, we demonstrate that infectious particles are present both within the infected cells and in the supernatant. Kinetic analysis indicates that intracellular particles constitute precursors of the secreted infectious virus. Ultracentrifugation analyses indicate that intracellular infectious viral particles are similar in size (∼65 to 70 nm) but different in buoyant density (∼1.15 to 1.20 g/ml) from extracellular particles (∼1.03 to 1.16 g/ml). These results indicate that infectious HCV particles are assembled intracellularly and that their biochemical composition is altered during viral egress.

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