Endocannabinoid CB1 antagonists inhibit hepatitis C virus production, providing a novel class of antiviral host-targeting agents

Direct-acting antivirals have significantly improved treatment outcomes in chronic hepatitis C (CHC), but side effects, drug resistance and cost mean that better treatments are still needed. Lipid metabolism is closely linked with hepatitis C virus (HCV) replication, and endocannabinoids are major regulators of lipid homeostasis. The cannabinoid 1 (CB1) receptor mediates these effects in the liver. We have previously shown upregulation of CB1 receptors in the livers of patients with CHC, and in a HCV cell-culture model. Here, we investigated whether CB1 blockade inhibited HCV replication. The antiviral effect of a CB1 antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), was examined in HCV strain JFH1 cell-culture and subgenomic replicon models. The effects on the expression of genes involved in lipid metabolism were also measured. CB1 short hairpin RNA (shRNA) was used to confirm that the effects were specific for the cannabinoid receptor. Treatment with AM251 strongly inhibited HCV RNA (~70 %), viral protein (~80 %), the production of new virus particles (~70 %) and virus infectivity (~90 %). As expected, AM251 reduced the expression of pro-lipogenic genes (SREBP-1c, FASN, SCD1 and ACC1) and stimulated genes promoting lipid oxidation (CPT1 and PPARα). This effect was mediated by AMP-activated protein kinase (AMPK). Stable CB1 knockdown of cells infected with HCV showed reduced levels of HCV RNA compared with controls. Thus, reduced CB1 signalling inhibits HCV replication using either pharmacological inhibitors or CB1 shRNA. This may be due, at least in part, to reduced lipogenesis, mediated by AMPK activation. We suggest that CB1 antagonists may represent an entirely new class of drug with activity against HCV.

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Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon

Journal of General Virology ◽

10.1099/vir.0.80006-0 ◽

2004 ◽

Vol 85 (7) ◽

pp. 1867-1875 ◽

Cited By ~ 10

Author(s):

Rita Graziani ◽

Giacomo Paonessa

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Hcv Rna ◽

Wild Type ◽

Subgenomic Replicon ◽

Adaptive Mutations ◽

Negative Effect

An efficient model is currently used to study hepatitis C virus (HCV) replication in cell culture. It involves transfection in Huh7, a hepatoma-derived cell line, of an antibiotic (neomycin) selectable HCV subgenomic replicon encoding the non-structural (NS) proteins from NS3 to NS5B. However, strong and sustained replication is achieved only on the appearance of adaptive mutations in viral proteins. The most effective of these adaptive mutations are concentrated mainly in NS5A, not only into the original Con1 but also in the recently established HCV-BK and HCV-H77 isolate-derived replicons. This suggests that the expression of wild-type (wt) NS5A may not allow efficient HCV RNA replication in cell culture. With the use of a β-lactamase reporter gene as a marker for HCV replication and TaqMan RNA analysis, the replication of different HCV replicons in cotransfection experiments was investigated. Comparing wt with NS5A-adapted replicons, the strong evidence accumulated showed that the expression of wt NS5A was actually able to inhibit the replication of NS5A-adapted replicons. This feature was characterized as a dominant negative effect. Interestingly, an NS5B (R2884G)-adapted replicon, containing a wt NS5A, was dominant negative on an NS5A-adapted replicon but was not inhibited by the original Con1 replicon. In conclusion, these studies revealed that the original wt Con1 replicon is not only incompetent for replication in cell culture, but is also able to interfere with NS5A-adapted replicons.

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Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013455 ◽

2020 ◽

Vol 295 (35) ◽

pp. 12426-12436 ◽

Cited By ~ 1

Author(s):

Lorillee Tallorin ◽

Valerie A. Villareal ◽

Chih-Yun Hsia ◽

Mary A. Rodgers ◽

Dominique J. Burri ◽

...

Keyword(s):

Hepatitis C Virus ◽

Lipid Metabolism ◽

Hepatitis C ◽

Molecular Mechanisms ◽

Rna Replication ◽

Hcv Rna ◽

Genome Replication ◽

Subgenomic Replicon ◽

Lipid Environment

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

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Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

Journal of Virology ◽

10.1128/jvi.00526-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 10999-11009 ◽

Cited By ~ 149

Author(s):

Pablo Gastaminza ◽

Kelly A. Dryden ◽

Bryan Boyd ◽

Malcolm R. Wood ◽

Mansun Law ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Structural Characteristics ◽

Buoyant Density ◽

Hcv Rna ◽

Membrane Bilayer ◽

Biophysical Characterization ◽

Negative Stain ◽

A Minor

ABSTRACT We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

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Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines

Journal of General Virology ◽

10.1099/vir.0.040725-0 ◽

2012 ◽

Vol 93 (7) ◽

pp. 1422-1431 ◽

Cited By ~ 8

Author(s):

Midori Takeda ◽

Masanori Ikeda ◽

Yasuo Ariumi ◽

Takaji Wakita ◽

Nobuyuki Kato

Keyword(s):

Cell Culture ◽

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Hepatoma Cell ◽

Rna Replication ◽

Hcv Rna ◽

Reporter Assay ◽

Hepatoma Cell Lines

A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann–Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication.

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Palmitoylation and Polymerization of Hepatitis C Virus NS4B Protein

Journal of Virology ◽

10.1128/jvi.00053-06 ◽

2006 ◽

Vol 80 (12) ◽

pp. 6013-6023 ◽

Cited By ~ 86

Author(s):

Guann-Yi Yu ◽

Ki-Jeong Lee ◽

Lu Gao ◽

Michael M. C. Lai

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Membrane Structure ◽

Rna Replication ◽

Polymerization Process ◽

Cysteine Residues ◽

Hcv Rna ◽

Subgenomic Replicon ◽

Lipid Modifications ◽

Ns4b Protein

ABSTRACT Hepatitis C Virus (HCV) NS4B protein induces a specialized membrane structure which may serve as the replication platform for HCV RNA replication. In the present study, we demonstrated that NS4B has lipid modifications (palmitoylation) on two cysteine residues (cysteines 257 and 261) at the C-terminal end. Site-specific mutagenesis of these cysteine residues on individual NS4B proteins and on an HCV subgenomic replicon showed that the lipid modifications, particularly of Cys261, are important for protein-protein interaction in the formation of the HCV RNA replication complex. We further demonstrated that NS4B can undergo polymerization. The main polymerization determinants were mapped in the N-terminal cytosolic domain of NS4B protein; however, the lipid modifications on the C terminus also facilitate the polymerization process. The lipid modification and the polymerization activity could be two properties of NS4B important for its induction of the specialized membrane structure involved in viral RNA replication.

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Hydrophobic and Charged Residues in the C-Terminal Arm of Hepatitis C Virus RNA-Dependent RNA Polymerase Regulate Initiation and Elongation

Journal of Virology ◽

10.1128/jvi.01106-14 ◽

2014 ◽

Vol 89 (4) ◽

pp. 2052-2063 ◽

Cited By ~ 5

Author(s):

Amy L. Cherry ◽

Caitriona A. Dennis ◽

Andrew Baron ◽

Leslie E. Eisele ◽

Pia A. Thommes ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

De Novo ◽

Structural Features ◽

Primer Extension ◽

Hcv Rna ◽

Genome Replication ◽

Rna Dependent Rna Polymerase ◽

Subgenomic Replicon

ABSTRACTThe RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a β-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of β-loop and C-terminal arm mutants, which were used forin vitroanalysis of RdRpde novoinitiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the β-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general.IMPORTANCEHCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr→Phe or Asp→Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

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Hepatitis C virus promotes virion secretion through cleavage of the Rab7 adaptor protein RILP

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1607277113 ◽

2016 ◽

Vol 113 (44) ◽

pp. 12484-12489 ◽

Cited By ~ 15

Author(s):

Ann L. Wozniak ◽

Abby Long ◽

Kellyann N. Jones-Jamtgaard ◽

Steven A. Weinman

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Time Course ◽

Rna Virus ◽

Adaptor Protein ◽

Virus Production ◽

Hcv Rna ◽

Cell Periphery ◽

Dynein Motor ◽

Intracellular Virus

Hepatitis C virus (HCV) is an enveloped RNA virus that modifies intracellular trafficking processes. The mechanisms that HCV and other viruses use to modify these events are poorly understood. In this study, we observed that two different RNA viruses, HCV and Sendai, cause inhibition of ras-related protein Rab-7 (Rab7)-dependent endosome–lysosome fusion. In both cases, viral infection causes cleavage of the Rab7 adaptor protein RILP (Rab interacting lysosomal protein), which is responsible for linking Rab7 vesicles to dynein motor complexes. RILP cleavage results in the generation of a cleaved RILP fragment (cRILP) missing the N terminus of the molecule. Although RILP localizes in a perinuclear fashion, cRILP moves to the cell periphery. Both knockdown of RILP and expression of cRILP reproduced the HCV-induced trafficking defect, and restoring full-length RILP reversed the trafficking effects of virus. For the first 3 d after electroporation of HCV RNA, intracellular virus predominates over secreted virus, but the quantity of intracellular virus then rapidly declines as secreted virus dominates. The transition from the intracellular-predominant to the secretion-predominant phenotype corresponds to the time course of cRILP generation. Expressing cRILP directly prevents intracellular virus accumulation at early times without affecting net virus production. The ability of cRILP to promote virus secretion could be prevented by a kinesin inhibitor. HCV thus modifies cellular trafficking by cleaving RILP, which serves to redirect Rab7-containing vesicles to a kinesin-dependent trafficking mode promoting virion secretion. Cleavage of a Rab adaptor protein is thus a mechanism by which viruses modify trafficking patterns of infected cells.

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Mutations in NS5B Polymerase of Hepatitis C Virus: Impacts on in Vitro Enzymatic Activity and Viral RNA Replication in the Subgenomic Replicon Cell Culture

Virology ◽

10.1006/viro.2002.1461 ◽

2002 ◽

Vol 297 (2) ◽

pp. 298-306 ◽

Cited By ~ 35

Author(s):

I.Wayne Cheney ◽

Suhaila Naim ◽

Vicky C.H. Lai ◽

Shannon Dempsey ◽

Daniel Bellows ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Enzymatic Activity ◽

Rna Replication ◽

Viral Rna ◽

Subgenomic Replicon ◽

Replicon Cell ◽

Ns5b Polymerase

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Participation of Rab5, an Early Endosome Protein, in Hepatitis C Virus RNA Replication Machinery

Journal of Virology ◽

10.1128/jvi.01366-06 ◽

2007 ◽

Vol 81 (9) ◽

pp. 4551-4563 ◽

Cited By ~ 88

Author(s):

Michelle Stone ◽

Shuaizheng Jia ◽

Won Do Heo ◽

Tobias Meyer ◽

Kouacou V. Konan

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Fluorescent Protein ◽

Early Endosome ◽

Dominant Negative ◽

Hcv Rna ◽

Small Interfering Rnas ◽

Subgenomic Replicon ◽

Gfp Fluorescence ◽

Positive Strand Rna

ABSTRACT Like most positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its genome on the surface of rearranged membranes. We have shown previously that HCV NS4AB, but not the product NS4B, inhibits endoplasmic reticulum (ER)-to-Golgi protein traffic (K. V. Konan, T. H. Giddings, Jr., M. Ikeda, K. Li, S. M. Lemon, and K. Kirkegaard, J. Virol. 77:7843-7855). However, both NS4AB and NS4B can induce “membranous web” formation, first reported by Egger et al. (D. B Egger, R. Gosert, L. Bianchi, H. E. Blum, D. Moradpour, and K. Bienz, J. Virol. 76:5974-5984), which is also observed in HCV-infected cells (Y. Rouille, F. Helle, D. Delgrange, P. Roingeard, C. Voisset, E. Blanchard, S. Belouzard, J. McKeating, A. H. Patel, G. Maertens, T. Wakita, C. Wychowski, and J. Dubuisson, J. Virol. 80:2832-2841) and cells that bear a subgenomic NS5A-green fluorescent protein (GFP) replicon (D. Moradpour, M. J. Evans, R. Gosert, Z. Yuan, H. E. Blum, S. P. Goff, B. D. Lindenbach, and C. M. Rice, J. Virol. 78:7400-7409). To determine the intracellular origin of the web, we examined NS4B colocalization with endogenous cellular markers in the context of the full-length or subgenomic replicon. We found that, in addition to ER markers, early endosome (EE) proteins, including Rab5, were associated with web-inducing protein NS4B. Furthermore, an immunoisolated fraction containing NS4B was found to contain both ER and EE proteins. Using fluorescence microscopy, we showed that wild-type and constitutively active Rab5 proteins were associated with NS4B. Interestingly, expression of dominant-negative Rab5 resulted in significant loss of GFP fluorescence in NS5A-GFP replicon cells. We also found that a small reduction in Rab5 protein expression decreased HCV RNA synthesis significantly. Furthermore, transfection of labeled Rab5 small interfering RNAs into NS5A-GFP replicon cells resulted in a significant decrease in GFP fluorescence. Finally, Rab5 protein was found to coimmunoprecipitate with HCV NS4B. These studies suggest that EE proteins, including Rab5, may play a role in HCV genome replication or web formation.

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Targeting Autophagy Augments Berberine-Mediated Cell Death in Human Hepatoma Cells Harboring Hepatitis C Virus RNA

Cells ◽

10.3390/cells9040908 ◽

2020 ◽

Vol 9 (4) ◽

pp. 908 ◽

Cited By ~ 1

Author(s):

Chen-Jei Tai ◽

Alagie Jassey ◽

Ching-Hsuan Liu ◽

Cheng-Jeng Tai ◽

Christopher D. Richardson ◽

...

Keyword(s):

Cell Death ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Hepatoma Cell ◽

Treatment Strategies ◽

Hepatoma Cells ◽

Hcv Rna ◽

Human Hepatoma Cells ◽

Subgenomic Replicon ◽

Pharmacological Inhibition

Hepatocellular carcinoma (HCC), including hepatitis C virus (HCV)-induced HCC, is a deadly disease highly refractory to chemotherapy, thus requiring the continuous identification of novel treatment strategies. Berberine (BBR) has been previously reported to inhibit hepatoma cell growth, but the main type of cell death elicited by BBR, and whether the alkaloid can inhibit hepatoma cells carrying HCV genomes, is unclear. Herein, we show that BBR treatment induced a biphasic cell death irrespective of the presence of HCV subgenomic replicon RNA, first triggering apoptosis that then progressed to necrosis between 24 and 48 h post-treatment. Furthermore, BBR treatment potentiated the HCV replicon-induced reactive oxygen species (ROS) production, inhibition of which with an antioxidant attenuated the cell death that was elicited by BBR in these cells. Moreover, BBR dampened the autophagic response in HCV RNA-positive or negative hepatoma cells, and pharmacological inhibition of autophagy conversely augmented the BBR-induced cell death. Finally, BBR inhibited the growth of Huh-7 cells that were persistently infected with the full-length genome HCV particles, and concomitant pharmacological inhibition of autophagy potentiated the killing of these cells by BBR. Our findings suggest that combining BBR with the inhibition of autophagy could be an attractive treatment strategy against HCC, irrespective of the presence of the HCV genome.

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