• Title/Summary/Keyword: HCV replicon

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Inhibition of the Replication of Hepatitis C Virus Replicon with Nuclease-Resistant RNA Aptamers

  • Shin, Kyung-Sook;Lim, Jong-Hoon;Kim, Jung-Hye;Myung, Hee-Joon;Lee, Seong-Wook
    • Journal of Microbiology and Biotechnology
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    • v.16 no.10
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    • pp.1634-1639
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    • 2006
  • Hepatitis C virus (HCV)-encoded nonstructural protein 5B (NS5B) possesses RNA-dependent RNA polymerase activity, which is considered essential for viral proliferation. Thus, HCV NS5B is a good therapeutic target protein for the development of anti-HCV agents. In this study, we isolated two different kinds of nuclease-resistant RNA aptamers with 2'-fluoro pyrimidines against the HCV NS5B from a combinatorial RNA library with 40 nucleotide random sequences, using SELEX technology. The isolated RNA aptamers were observed to specifically and avidly bind the HCV NS5B with an apparent $K_d$ of 5 nM and 18 nM, respectively, in contrast with the original RNA library that hardly bound the target protein. Moreover, these aptamers could partially inhibit RNA synthesis of the HCV subgenomic replicon when transfected into Huh-7 hepatoma cell lines. These results suggest that the RNA aptamers selected in vitro could be useful not only as therapeutic agents of HCV infection but also as a powerful tool for the study of the HCV RNA-dependent RNA polymerase mechanism.

Inhibition of Hepatitis C Virus (HCV) Replication by Hammerhead Ribozyme Which Activity Can Be Allosterically Regulated by HCV NS5B RNA Replicase (C형 간염바이러스(HCV)의 NS5B RNA Replicase에 의해 활성이 유도되는 Hammerhead 리보자임에 의한 HCV 복제 억제 연구)

  • Lee, Chang-Ho;Lee, Seong-Wook
    • Korean Journal of Microbiology
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    • v.47 no.3
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    • pp.188-193
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    • 2011
  • As a specific and effective therapeutic genetic material against hepatitis C virus (HCV) multiplication, HCV internal ribosome entry site (IRES)-targeting hammerhead ribozyme which activity is allosterically regulated by HCV regulatory protein, NS5B RNA replicase, was constructed. The allosteric ribozyme was composed of sequence of RNA aptamer to HCV NS5B, communication module sequence which can transfer structural transition for inducing ribozyme activity upon binding NS5B to the aptamer, and sequence of ribozyme targeting +382 nucleotide of HCV IRES. With real-time PCR analysis, the ribozyme was found to efficiently inhibit HCV replicon replication in cells. Of note, the allosteric ribozyme was shown to inhibit HCV replicon replication more efficiently than either HCV genome-targeting ribozyme or NS5B aptamer only. This allosteric ribozyme can be used as a lead genetic agent for the specific and effective suppression of HCV replication.

Analysis of In Vivo Interaction of HCV NS3 Protein and Specific RNA Aptamer with Yeast Three-Hybrid System

  • HWANG BYOUNGHOON;LEE SEONG-WOOK
    • Journal of Microbiology and Biotechnology
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    • v.15 no.3
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    • pp.660-664
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    • 2005
  • We have previously isolated specific RNA aptamers with high affinity against the helicase domain of hepatitis C virus (HCV) nonstructural protein 3 (NS3). The RNA aptamers competitively and efficiently inhibited the helicase activity, partially impeding HCV replicon replication in human hepatocarcinoma cells. In this study, the RNA aptamers were tested for binding to the HCV NS3 proteins in eukaryotic cells, using a yeast three-hybrid system. The aptamers were then recognized by the HCV NS3 proteins when expressed in the cells, while the antisense sequences of the aptamers were not. These results suggest that the in vitro selected RNA aptamers can also specifically bind to the target proteins in vivo. Consequently, they could be potentially utilized as anti-HCV lead compounds.

2'-Spirocyclopropyl-carbocyclic Nucleoside as a Novel Scaffold for Potent Anti-HCV Agents

  • Li, Hua;Yoo, Jin-Cheol;Hong, Joon-Hee
    • Bulletin of the Korean Chemical Society
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    • v.32 no.4
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    • pp.1146-1152
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    • 2011
  • The discovery of 2'-spirocyclopropyl-ribocytidine (J. Med. Chem. 2010, 53, 8150-8160) as a potent inhibitor of RNA synthesis by NS5B ($IC_{50}=7.3{\mu}M$), the RNA polymerase encoded by hepatitis C Virus (HCV), has led to the synthesis and biological evaluation of several carbocyclic versions of 2'-spiropropyl-nucleosides. The cyclopentenol intermediate 7 was successfully constructed via ring-closing metathesis (RCM) from divinyl 6. Spirocyclopropanation of enone 8 was effected by using (2-chloroethyl)-dimethylsulfonium iodide and potassium tert-butoxide to form the desired intermediate 9. The synthesized nucleoside analogues 21-24 were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line. Among them, the cytosine nucleoside analogue 22 exhibited significant anti-HCV activity ($EC_{50}= 8.2{\mu}M$).

Synthesis and Anti-HCV Activity of 3',5'-cyclic SATE Phosphonodiester Nucleoside as a Novel Prodrug

  • Liu, Lian Jin;Seo, Rac-Seok;Yoo, Seung-Won;Choi, Jin;Hong, Joon-Hee
    • Bulletin of the Korean Chemical Society
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    • v.31 no.4
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    • pp.915-920
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    • 2010
  • A novel 2',4'-dimethyl carbocyclic adenosine 5'-phosphonic acid analogue (20) was prepared using acyclic stereoselective route from commercially available 4-hydroxybutan-2-one (4). To improve cellular permeability and enhance the anti-HCV activity of this phosphonic acid, a 3',5'-cyclic SATE phosphonodiester nucleoside prodrug (22) was prepared. The synthesized phosphonic nucleoside analogues, (20) and (22), were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line.

Synthesis and Antiviral Activity of 2'(β)-Hydroxymethylated Carbodine Analogues Against Hepatitis C Virus

  • Hong, Joon-Hee;Oh, Chang-Hyun
    • Bulletin of the Korean Chemical Society
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    • v.30 no.11
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    • pp.2626-2630
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    • 2009
  • 2'($\beta$)-Hydroxymethylated adenosine is a potent and selective inhibitor of hepatitis C virus (HCV) replication. It targets the RNA-dependent RNA polymerase of HCV, NS5B. Synthesis and antiviral evaluation of carbocyclic versions are described. The cyclopentene intermediate ($9\beta$) was successfully synthesized through sequential Johnson-Claisen orthoester rearrangement and ring-closing metathesis (RCM). Coupling of bases via a Pd(0) catalyst, selective dihydroxylation, and desilylation yielded the target nucleoside analogues. The compounds 17 and 18 were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line and showed moderate antiviral activity with toxicity up to 20.0 and 24.7 ${\mu}g/mL$, respectively.

Interaction of Stomatin with Hepatitis C Virus RNA Polymerase Stabilizes the Viral RNA Replicase Complexes on Detergent-Resistant Membranes

  • Kim, Jung-Hee;Rhee, Jin-Kyu;Ahn, Dae-Gyun;Kim, Kwang Pyo;Oh, Jong-Won
    • Journal of Microbiology and Biotechnology
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    • v.24 no.12
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    • pp.1744-1754
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    • 2014
  • The hepatitis C virus (HCV) RNA genome is replicated by an RNA replicase complex (RC) consisting of cellular proteins and viral nonstructural (NS) proteins, including NS5B, an RNA-dependent RNA polymerase (RdRp) and key enzyme for viral RNA genome replication. The HCV RC is known to be associated with an intracellular membrane structure, but the cellular components of the RC and their roles in the formation of the HCV RC have not been well characterized. In this study, we took a proteomic approach to identify stomatin, a member of the integral proteins of lipid rafts, as a cellular protein interacting with HCV NS5B. Co-immunoprecipitation and co-localization studies confirmed the interaction between stomatin and NS5B. We demonstrated that the subcellular fraction containing viral NS proteins and stomatin displays RdRp activity. Membrane flotation assays with the HCV genome replication-competent subcellular fraction revealed that the HCV RdRp and stomatin are associated with the lipid raft-like domain of membranous structures. Stomatin silencing by RNA interference led to the release of NS5B from the detergent-resistant membrane, thereby inhibiting HCV replication in both HCV subgenomic replicon-harboring cells and HCV-infected cells. Our results identify stomatin as a cellular protein that plays a role in the formation of an enzymatically active HCV RC on a detergent-resistant membrane structure.

Evaluation of Inhibitory Effects of Thiobarbituric Acid Derivatives Targeting HCV NS5B Polymerase

  • Lee, Jong-Ho;Lee, Sang-Yoon;Park, Mi-Young;Ha, Hyun-Joon;Myung, Hee-Joon
    • Journal of Microbiology and Biotechnology
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    • v.20 no.3
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    • pp.510-512
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    • 2010
  • A series of thiobarbituric acid derivatives were constructed and evaluated for inhibitory activity on hepatitis C virus NS5B polymerase. In biochemical assays using purified viral polymerase and RNA template, the $IC_{50}$ value was improved to 0.41 ${\mu}M$ from the original compound's 1.7 ${\mu}M$ value. In HCV sub genomic replicon assay, the $EC_{50}$ value was improved to 3.7 ${\mu}M$ from the original compound's 12.3 ${\mu}M$ value. $CC_{50}$ was higher than 77 ${\mu}M$ for all compounds tested, suggesting that they are useful candidates for anti-HCV therapy.

Purification and Characterization of HCV RNA-dependent RNA Polymerase from Korean Genotype 1b Isolate: Implications for Discovery of HCV Polymerase Inhibitors

  • Kim, Jeong-Min;Lee, Mi-Kyoung;Kim, Yong-Zu
    • Bulletin of the Korean Chemical Society
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    • v.26 no.2
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    • pp.285-291
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    • 2005
  • The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is the viral RNA-dependent RNA polymerase (RdRp), which is the essential catalytic enzyme for the viral replication and is an appealing target for the development of new therapeutic agents against HCV infection. A small amount of serum from a single patient with hepatitis C was used to get the genome of a Korean HCV isolate. Sequence analysis of NS5B 1701 nucleotides showed the genotype of a Korean isolate to be subtype 1b. The soluble recombinant HCV NS5B polymerase lacking the C-terminal 24 amino acids was expressed and purified to homogeneity. With the highly purified NS5B protein, we established in vitro systems for RdRp activity to identify potential polymerase inhibitors. The rhodanine family compounds were found to be potent and specific inhibitors of NS5B from high throughput screening (HTS) assay utilizing the scintillation proximity assay (SPA) system. The binding mode of an inhibitor was analyzed by measuring various kinetic parameters. Lineweaver-Burk plots of the inhibitor suggested it binds not to the active site of NS5B polymerase, but to an allosteric site of the enzyme. The activity of NS5B in in vitro polymerase reactions with homopolymeric RNA requires interaction with multiple substrates that include a template/primer and ribonucleotide triphosphate. Steady-state kinetic parameter, such as Km, was determined for the ribonucleotide triphosphate. One of compounds found interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitively with respect to UTP. Furthermore, we also investigated the ability of the compound to inhibit NS5B-directed viral RNA replication using the Huh7 cell-based HCV replicon system. The investigation is potentially very useful for the utility of such compounds as anti-hepatitic agents.