Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture

ABSTRACT A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 Å away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 μM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.

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

Journal of General Virology ◽

10.1099/vir.0.067231-0 ◽

2014 ◽

Vol 95 (11) ◽

pp. 2468-2479 ◽

Cited By ~ 10

Author(s):

Mahsa Shahidi ◽

Enoch S. E. Tay ◽

Scott A. Read ◽

Mehdi Ramezani-Moghadam ◽

Kazuaki Chayama ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Lipid Metabolism ◽

Hepatitis C ◽

Cannabinoid Receptor ◽

Virus Production ◽

Hcv Rna ◽

Virus Infectivity ◽

Subgenomic Replicon ◽

Cb1 Antagonists

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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Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles

Journal of Viral Hepatitis ◽

10.1111/j.1365-2893.2009.01111.x ◽

2009 ◽

Vol 16 (9) ◽

pp. 621-632 ◽

Cited By ~ 26

Author(s):

R. S. Russell ◽

K. Kawaguchi ◽

J.-C. Meunier ◽

S. Takikawa ◽

K. Faulk ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Infectious Virus ◽

Mutational Analysis ◽

Virus Particles

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Replicon Cell Culture System as a Valuable Tool in Antiviral Drug Discovery against Hepatitis C Virus

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020501600101 ◽

2005 ◽

Vol 16 (1) ◽

pp. 1-12 ◽

Cited By ~ 28

Author(s):

Nigel Horscroft ◽

Vicky CH Lai ◽

Wayne Cheney ◽

Nanhua Yao ◽

Jim Z Wu ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Drug Discovery ◽

Culture System ◽

Antiviral Drug ◽

Small Animal ◽

Hcv Rna ◽

Cell Culture System ◽

Replicon Cell

Discovery of potential therapeutics against hepatitis C virus (HCV) infection has been hampered in the past decade by the inability to grow this virus in tissue culture and by the lack of robust small animal models. This situation has been improved by the recent development of a selectable HCV replicon cell culture system. For the first time, drug discovery scientists are able to screen large compound collections using the replicon cell culture system to identify small molecules with the potential to inhibit HCV RNA replication. The replicon system has also been used to elucidate inhibitors' antiviral mechanism of action and to optimize antiviral potency. In this review, we will summarize the recent development of HCV replicon cell culture system and its use in anti-HCV drug discovery. The antiviral activities of promising lead compounds are also reviewed.

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