Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

Ma, Han; Jiang, Wen-Rong; Robledo, Nicole; Lévêque, Vincent; Ali, Samir; Lara‐Jaime, Teresa; Masjedizadeh, Mohammad R.; Smith, David B.; Cammack, Nick; Klumpp, Klaus; Symons, Julian

doi:10.1074/jbc.m705274200

Cited by 107 publications

(118 citation statements)

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“…Because transfected RNA is likely to be subject to different decay pathways than replicating viral genomes in infected cells, we determined whether miR-122 also slows degradation of viral RNA in infected cells treated with PSI-6130, a potent and specific nucleoside inhibitor that arrests new viral RNA synthesis (18). Under these conditions, as expected, viral RNA degraded more slowly than after electroporation (compare Fig.…”

Section: Resultsmentioning

confidence: 91%

Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex

Shimakami

Yamane

Jangra

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

358

385

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MicroRNAs (miRNAs) are small noncoding RNAs that regulate eukaryotic gene expression by binding to regions of imperfect complementarity in mRNAs, typically in the 3′ UTR, recruiting an Argonaute (Ago) protein complex that usually results in translational repression or destabilization of the target RNA. The translation and decay of mRNAs are closely linked, competing processes, and whether the miRNA-induced silencing complex (RISC) acts primarily to reduce translation or stability of the mRNA remains controversial. miR-122 is an abundant, liver-specific miRNA that is an unusual host factor for hepatitis C virus (HCV), an important cause of liver disease in humans. Prior studies show that it binds the 5′ UTR of the messenger-sense HCV RNA genome, stimulating translation and promoting genome replication by an unknown mechanism. Here we show that miR-122 binds HCV RNA in association with Ago2 and that this slows decay of the viral genome in infected cells. The stabilizing action of miR-122 does not require the viral RNA to be translationally active nor engaged in replication, and can be functionally substituted by a nonmethylated 5′ cap. Our data demonstrate that a RISC-like complex mediates the stability of HCV RNA and suggest that Ago2 and miR-122 act coordinately to protect the viral genome from 5′ exonuclease activity of the host mRNA decay machinery. miR-122 thus acts in an unconventional fashion to stabilize HCV RNA and slow its decay, expanding the repertoire of mechanisms by which miRNAs modulate gene expression.

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Section: Resultsmentioning

confidence: 91%

Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex

Shimakami

Yamane

Jangra

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

358

385

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“…3C). At 24 h after removal of extracellular RO-9187, the intracellular RO-9187-TP concentrations were still well above 4 pmol/10 6 (1.3 M) cells, and thus above the antiviral IC 50 , assuming a 3-l volume of normal human liver parenchymal cells (20,21), reflective of very robust phosphorylation efficiency of RO-9187 in primary human hepatocytes.…”

Section: Ro-9187 Is Efficiently Phosphorylated In Primary Humanmentioning

confidence: 94%

“…Analysis of Phosphorylation in Primary Human HepatocytesNucleoside phosphorylation efficiency and the intracellular half-life of the 5Ј-triphosphate of RO-9187 were determined by incubating fresh primary human hepatocytes with tritium-labeled RO-9187 as described (20) with the following modifications of the cell extraction procedure. The soluble content of the primary hepatocytes was incubated with 500 l of 1.5 M perchloric acid for 30 min on ice, followed by neutralization with 300 l of 2.5 N potassium hydroxide.…”

Section: Methodsmentioning

confidence: 99%

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2′-Deoxy-4′-azido Nucleoside Analogs Are Highly Potent Inhibitors of Hepatitis C Virus Replication Despite the Lack of 2′-α-Hydroxyl Groups

Klumpp

Kalayanov

et al. 2008

Journal of Biological Chemistry

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RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2-␣-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of ␣-hydroxy moieties. 2-Deoxy-2-␤-fluoro-4-azidocytidine (RO-0622) and 2-deoxy-2-␤-hydroxy-4-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC 50 ‫؍‬ 171 ؎ 12 nM and 24 ؎ 3 nM for RO-9187 and RO-0622, respectively; CC 50 >1 mM for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4-azidocytidine) or 2-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC 50 values 8 -150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2-␣-deoxy-4-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection. Hepatitis C virus (HCV)3 infection is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation. Current treatment options available to HCV-infected persons have limitations with regard to efficacy and tolerability. Only about 50% of individuals infected with HCV genotype 1 achieve sustained virological response when treated with a combination of pegylated interferon ␣ and ribavirin (1, 2). In addition, high viral load, age, body weight, co-infection with human immunodeficiency virus, and cirrhosis negatively affect the probability of achieving sustained virological response (3, 4). Therefore, there is an urgent need to develop new and more effective therapies for the treatment of HCV infection. A number of new antiviral candidates are currently being evaluated in clinical studies, the majority targeting either the HCV protease or HCV polymerase enzymes, which are essential for viral replication (5). The HCV RNA-dependent RNA polymerase, NS5B, contains the active site responsible for viral RNA synthesis and functions as part of a membrane-associated replicase complex. Nucleoside and non-nucleoside inhibitors of HCV polymerase h...

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“…Indeed, significant differences have been noted between different hepatoma cell lines as well as between hepatoma cell lines and primary human hepatocytes (Hernandez-Santiago et al, 2004;Klumpp et al, 2008;Paeshuyse et al, 2008). Interestingly, it has been reported that the phosphorylation of some nucleoside analog derivatives that is observed in primary human hepatocytes does not occur in replicon cells (Huh-7) either because the compounds cannot enter the cells or because of a deficient conversion (Ma et al, 2007). This clearly emphasizes the risk of false negative in the screening of anti-HCV compounds using solely the replicon systems.…”

Section: Triyatni Et Al Observed No Inhibition Of Hcv-lp Entry Intomentioning

confidence: 99%

Cellular models for the screening and development of anti-hepatitis C virus agents

Gondeau

Pichard-Garcia

Maurel

2009

Pharmacology & Therapeutics

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Investigations on the biology of hepatitis C virus (HCV) have been hampered by the lack of small animal models. Efforts have therefore been directed to designing practical and robust cellular models of human origin able to support HCV replication and production in a reproducible, reliable and consistent manner. Many different models based on different forms of virions and hepatoma or other cell types have been described including virus-like particles, pseudotyped particles, subgenomic and full length replicons, virion productive replicons, immortalised hepatocytes, fetal and adult primary human hepatocytes. This review focuses on these different cellular models, their advantages and disadvantages at the biological and experimental levels, and their respective use for evaluating the effect of antiviral molecules on different steps of HCV biology including virus entry, replication, particles generation and excretion, as well as on the modulation by the virus of the host cell response to infection.

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Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

Cited by 107 publications

References 22 publications

Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex

Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex

2′-Deoxy-4′-azido Nucleoside Analogs Are Highly Potent Inhibitors of Hepatitis C Virus Replication Despite the Lack of 2′-α-Hydroxyl Groups

Cellular models for the screening and development of anti-hepatitis C virus agents

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