Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells

Bellecave, Pantxika; Cazenave, Christian; Rumi, Julie; Staedel, Cathy; Cosnefroy, Ophélie; Andréola, Marie‐Line; Ventura, Michel; Tarragó-Litvak, Laura; Astier-Gin, T.

doi:10.1128/aac.01227-07

Cited by 41 publications

(39 citation statements)

References 50 publications

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“…It seems possible that the inhibitory effect of AO-01 was relatively strong during the early phase after the transfection, while later during the 3-day period, virus production may be more and more undisturbed. A DNA aptamer against the NSB5 protein of hepatitis C virus has a dissociation constant of 132 nM, which is similar to the K d of aptamer AO-01, and reduced viral mRNA levels by 90% in a cell culture system (32). Another possible explanation besides the short half-life for the relatively weak inhibition of budding by aptamer AO-01 is that the MBD of mature HBV capsids competent for budding may have a structure different from that of the MBD of recombinant capsids lacking the C-terminal domain.…”

Section: Discussionmentioning

confidence: 99%

An Aptamer against the Matrix Binding Domain on the Hepatitis B Virus Capsid Impairs Virion Formation

Orabi

Bieringer

Geerlof

et al. 2015

J Virol

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The hepatitis B virus (HBV Hepatitis B virus (HBV) has infected more than 40% of the living human population and causes 240 million persistent infections (1). The treatment of chronic infections is limited to date to use of inhibitors of the viral reverse transcriptase and stimulation of the immune system by interferon. Alternative antiviral strategies are desirable.During HBV replication, an RNA molecule (pregenome) is packaged together with the reverse transcriptase by 180 or 240 copies of the viral core protein. The assembled particle is an icosahedron with T ϭ 3 or T ϭ 4 symmetry and has a diameter of approximately 30 nm. The pregenome serves as a template for the synthesis of the viral DNA genome by reverse transcription occurring in the lumen of the capsid (2). This particle can then be enveloped by the three viral transmembrane surface proteins S, M, and L at an intracellular membrane, and the resulting virion is subsequently secreted from the host cell (3). Interestingly, the immature capsid containing the pregenome is not enveloped, in contrast to the case for the mature, DNA-containing capsid (4). Apparently, a structural change of the capsid surface is coupled to the synthesis of the viral DNA genome (5).Heterologous expression of the core protein in eukaryotic cells and even in bacteria leads to capsids almost indistinguishable from authentic capsids with respect to their antigenicity and appearance by electron microscopy. The C-terminal 30-amino-acid (aa)-long region of the core protein is very rich in arginine residues and has nucleic acid binding properties. Deletion of this domain is compatible with capsid formation (6).The budding of mature capsids is supported by cellular factors involved in multivesicular body formation (7). In addition, budding is dependent on a linear, 22-aa-long domain (matrix domain [MD]) of the surface protein L exposed at the cytoplasmic side of the cellular membrane and on a region on the capsid surface (matrix binding domain [MBD]) comprised of a ring-like groove around the base of the spike protruding from the capsid and a small area close to the pores of the capsid shell (8). Numerous single point mutations in either of the two domains block nucleocapsid envelopment (9, 10). It seems conceivable that the two

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Section: Discussionmentioning

confidence: 99%

An Aptamer against the Matrix Binding Domain on the Hepatitis B Virus Capsid Impairs Virion Formation

Orabi

Bieringer

Geerlof

et al. 2015

J Virol

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“…Moreover, their inhibitory effects against HCV should be carefully tested using the genotype 3a cell culture system that has been recently developed [76] . Bellecave et al [77] also reported DNA aptamers against HCV NS5B, and their aptamers inhibited HCV JFH-1 replication and viral particle formation in the cell culture system. However, those aptamers were not examined with regard to cell toxicity profiles, distribution in animals, or side effects during long-term treatment.…”

Section: Ns5b Targeting Aptamersmentioning

confidence: 94%

“…Hairpin ribozyme Cleave target RNAs 5'-UTR [24,28] , 3'-UTR [28] , and core region [24] Tested in in vitro [24] and in cell culture model [28] HDV ribozyme 5'-UTR [30] Tested in in vitro Hammerhead ribozyme (Hepatozyme) 5'-UTR [33][34][35] Completion of phase Ⅱ DNAzyme 5'-UTR [41] , core and NS5B region [39,40] Tested in in vitro and in cell culture model RNase P 5'-UTR [45][46][47] Splicing riboyme Selectively replace target RNAs with desirable RNAs 5'-UTR [51] Allosteric ribozyme Inhibit HCV replication [54] or cleave HCV RNA [31] through recognizing ligands miR-122 [54] and 5'-UTR [31] Aptamer Bind to target molecule and function as decoys and/or inhibitors NS3 [68][69][70] NS5B [75][76][77][78] E1E2 [79] Viral RNA [80,81] …”

Section: Mode Of Action Target Statusmentioning

confidence: 99%

Prospects for nucleic acid-based therapeutics against hepatitis C virus

Lee

Kim

Lee

2013

WJG

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In this review, we discuss recent advances in nucleic acid-based therapeutic technologies that target hepatitis C virus (HCV) infection. Because the HCV genome is present exclusively in RNA form during replication, various nucleic acid-based therapeutic approaches targeting the HCV genome, such as ribozymes, aptamers, siRNAs, and antisense oligonucleotides, have been suggested as potential tools against HCV. Nucleic acids are potentially immunogenic and typically require a delivery tool to be utilized as therapeutics. These limitations have hampered the clinical development of nucleic acid-based therapeutics. However, despite these limitations, nucleic acid-based therapeutics has clinical value due to their great specificity, easy and large-scale synthesis with chemical methods, and pharmaceutical flexibility. Moreover, nucleic acid therapeutics are expected to broaden the range of targetable molecules essential for the HCV replication cycle, and therefore they may prove to be more effective than existing therapeutics, such as interferon-α and ribavirin combination therapy. This review focuses on the current status and future prospects of ribozymes, aptamers, siRNAs, and antisense oligonucleotides as therapeutic reagents against HCV.

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“…Bellecave et al reported DNA aptamers that inhibit HCV JFH-1 replication and viral particle formation in a cell culture system (53). However, those DNA aptamers were not examined with regard to cell toxicity profiles, distribution in animals, or side effects during long-term treatment.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Hepatitis C Virus (HCV) Replication by Specific RNA Aptamers against HCV NS5B RNA Replicase

Lee

Kim

et al. 2013

J Virol

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This study identified specific and avid RNA aptamers consisting of 2=-hydroxyl-or 2=-fluoropyrimidines against hepatitis C virus (HCV) NS5B replicase, an enzyme that is essential for HCV replication. These aptamers acted as potent decoys to competitively impede replicase-catalyzed RNA synthesis activity. Cytoplasmic expression of the 2=-hydroxyl aptamer efficiently inhibited HCV replicon replication in human liver cells through specific interaction with, and sequestration of, the target protein without either off-target effects or escape mutant generation. A selected 2=-fluoro aptamer could be truncated to a chemically manufacturable length of 29 nucleotides (nt), with increase in the affinity to HCV NS5B. Noticeably, transfection of the truncated aptamer efficiently suppressed HCV replication in cells without escape mutant appearance. The aptamer was further modified through conjugation of a cholesterol or galactose-polyethylene glycol ligand for in vivo availability and liver-specific delivery. The conjugated aptamer efficiently entered cells and inhibited genotype 1b subgenomic and genotype 2a full-length HCV JFH-1 RNA replication without toxicity and innate immunity induction. Importantly, a therapeutically feasible amount of the conjugated aptamer was delivered in vivo to liver tissue in mice. Therefore, cytoplasmic expression of 2=-hydroxyl aptamer or direct administration of chemically synthesized and ligand-conjugated 2=-fluoro aptamer against HCV NS5B could be a potent anti-HCV approach. Hepatitis C virus (HCV) is the main causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1, 2). Although HCV infection causes worldwide health problems, efficient and specific antiviral therapy has not yet been developed.HCV belongs to the genus Hepacivirus in the family Flaviviridae. The enveloped virus contains a single, positive-stranded RNA genome about 9.6 kb in length encoding a single polyprotein of about 3,010 amino acids (3). This polyprotein precursor is co-or posttranslationally processed by cellular and viral proteases to yield functional structural and nonstructural proteins (4). The structural proteins include the core protein, C, and the envelope glycoproteins E1 and E2. The nonstructural (NS) proteins comprise the protease NS2, the multifunctional protein NS3 consisting of serine protease and helicase, the serine protease cofactor NS4A, the proteins NS4B and NS5A, and the RNA-dependent RNA polymerase NS5B, which are components of a complex responsible for HCV replication (5). NS5B is the central catalytic enzyme in HCV RNA replication. A number of drugs targeting HCV NS5B replicase are under development. However, the rapid appearance of drug-resistant escape mutant viruses has been reported (6).RNA aptamers are small structured single-stranded RNAs which have emerged as attractive and feasible alternatives to small-molecule and antibody-based therapy. Aptamers can be evolved by systematic evolution of ligands by exponential enrichment (SELEX), an iterative selection method...

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Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells

Cited by 41 publications

References 50 publications

An Aptamer against the Matrix Binding Domain on the Hepatitis B Virus Capsid Impairs Virion Formation

An Aptamer against the Matrix Binding Domain on the Hepatitis B Virus Capsid Impairs Virion Formation

Prospects for nucleic acid-based therapeutics against hepatitis C virus

Inhibition of Hepatitis C Virus (HCV) Replication by Specific RNA Aptamers against HCV NS5B RNA Replicase

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