scholarly journals Mechanism of Activation of β-d-2′-Deoxy-2′-Fluoro-2′-C-Methylcytidine and Inhibition of Hepatitis C Virus NS5B RNA Polymerase

2007 ◽  
Vol 51 (2) ◽  
pp. 503-509 ◽  
Author(s):  
Eisuke Murakami ◽  
Haiying Bao ◽  
Mangala Ramesh ◽  
Tamara R. McBrayer ◽  
Tony Whitaker ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Specific Inhibitor ◽  
Nucleoside Diphosphate Kinase ◽  
Hcv Rna ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Hcv Ns5b

ABSTRACT β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent specific inhibitor of hepatitis C virus (HCV) RNA synthesis in Huh-7 replicon cells. To inhibit the HCV NS5B RNA polymerase, PSI-6130 must be phosphorylated to the 5′-triphosphate form. The phosphorylation of PSI-6130 and inhibition of HCV NS5B were investigated. The phosphorylation of PSI-6130 by recombinant human 2′-deoxycytidine kinase (dCK) and uridine-cytidine kinase 1 (UCK-1) was measured by using a coupled spectrophotometric reaction. PSI-6130 was shown to be a substrate for purified dCK, with a Km of 81 μM and a k cat of 0.007 s−1, but was not a substrate for UCK-1. PSI-6130 monophosphate (PSI-6130-MP) was efficiently phosphorylated to the diphosphate and subsequently to the triphosphate by recombinant human UMP-CMP kinase and nucleoside diphosphate kinase, respectively. The inhibition of wild-type and mutated (S282T) HCV NS5B RNA polymerases was studied. The steady-state inhibition constant (Ki ) for PSI-6130 triphosphate (PSI-6130-TP) with the wild-type enzyme was 4.3 μM. Similar results were obtained with 2′-C-methyladenosine triphosphate (Ki = 1.5 μM) and 2′-C-methylcytidine triphosphate (Ki = 1.6 μM). NS5B with the S282T mutation, which is known to confer resistance to 2′-C-methyladenosine, was inhibited by PSI-6130-TP as efficiently as the wild type. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B RNA polymerase resulted in chain termination.

scholarly journals De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

2000 ◽  
Vol 74 (2) ◽  
pp. 851-863 ◽  
Author(s):  
Guangxiang Luo ◽  
Robert K. Hamatake ◽  
Danielle M. Mathis ◽  
Jason Racela ◽  
Karen L. Rigat ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Oligonucleotide Primer ◽  
Transferase Activity ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hcv Ns5b

ABSTRACT Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

scholarly journals 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

2008 ◽  
Vol 52 (6) ◽  
pp. 2097-2110 ◽  
Author(s):  
Pantxika Bellecave ◽  
Christian Cazenave ◽  
Julie Rumi ◽  
Cathy Staedel ◽  
Ophélie Cosnefroy ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Selection Procedure ◽  
Inhibitory Effect ◽  
Hcv Rna ◽  
Dna Aptamers ◽  
Huh7 Cells

ABSTRACT We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.

scholarly journals A Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase Capable of Copying the Full-Length Viral RNA

1999 ◽  
Vol 73 (9) ◽  
pp. 7694-7702 ◽  
Author(s):  
Jong-Won Oh ◽  
Takayoshi Ito ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Full Length ◽  
Viral Rna ◽  
Hcv Rna ◽  
Independent Manner ◽  
Virus Rna

ABSTRACT All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer-independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viral RNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3′-end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5′ end or 45 nt from the 3′ end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3′-end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3′ end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3′ end. These data defined the cis-acting sequences for HCV RNA synthesis at the 3′ end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.

scholarly journals General Catalytic Deficiency of Hepatitis C Virus RNA Polymerase with an S282T Mutation and Mutually Exclusive Resistance towards 2′-Modified Nucleotide Analogues

2006 ◽  
Vol 50 (12) ◽  
pp. 4161-4169 ◽  
Author(s):  
Hélène Dutartre ◽  
Cécile Bussetta ◽  
Joëlle Boretto ◽  
Bruno Canard
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Resistance Mutation ◽  
Viral Fitness ◽  
Hcv Rna ◽  
Phase Ii Trials ◽  
Nucleotide Analogues

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is an important target for antiviral therapies. NS5B is able to initiate viral RNA synthesis de novo and then switch to a fast and processive RNA elongation synthesis mode. The nucleotide analogue 2′-C-methyl CTP (2′-C-Me-CTP) is the active metabolite of NM283, a drug currently in clinical phase II trials. The resistance mutation S282T can be selected in HCV replicon studies. Likewise, 2′-O-Me nucleotides are active both against the purified polymerase and in replicon studies. We have determined the molecular mechanism by which the S282T mutation confers resistance to 2′-modified nucleotide analogues. 2′-C-Me-CTP is no longer incorporated during the initiation step of RNA synthesis and is discriminated 21-fold during RNA elongation by the NS5B S282T mutant. Strikingly, 2′-O-methyl CTP sensitivity does not change during initiation, but the analogue is no longer incorporated during elongation. This mutually exclusive resistance mechanism suggests not only that “2′-conformer” analogues target distinct steps in RNA synthesis but also that these analogues have interesting potential in combination therapies. In addition, the presence of the S282T mutation induces a general cost in terms of polymerase efficiency that may translate to decreased viral fitness: natural nucleotides become 5- to 20-fold less efficiently incorporated into RNA by the NS5B S282T mutant. As in the case for human immunodeficiency virus, our results might provide a mechanistic basis for the rational combination of drugs for low-fitness viruses.

scholarly journals Biochemical Study of the Comparative Inhibition of Hepatitis C Virus RNA Polymerase by VX-222 and Filibuvir

2011 ◽  
Vol 56 (2) ◽  
pp. 830-837 ◽  
Author(s):  
Guanghui Yi ◽  
Jerome Deval ◽  
Baochang Fan ◽  
Hui Cai ◽  
Charlotte Soulard ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Dissociation Constants ◽  
Biochemical Study ◽  
Hcv Rna ◽  
Resistance Mutations ◽  
Subgenomic Replicon

ABSTRACTFilibuvir and VX-222 are nonnucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of the hepatitis C virus (HCV) RNA-dependent RNA polymerase. Both compounds have shown significant promise in clinical trials and, therefore, it is relevant to better understand their mechanisms of inhibition. In our study, filibuvir and VX-222 inhibited the 1b/Con1 HCV subgenomic replicon, with 50% effective concentrations (EC50s) of 70 nM and 5 nM, respectively. Using several RNA templates in biochemical assays, we found that both compounds preferentially inhibited primer-dependent RNA synthesis but had either no or only modest effects onde novo-initiated RNA synthesis. Filibuvir and VX-222 bind to the HCV polymerase with dissociation constants of 29 and 17 nM, respectively. Three potential resistance mutations in the thumb II pocket were analyzed for effects on inhibition by the two compounds. The M423T substitution in the RNA polymerase was at least 100-fold more resistant to filibuvir in the subgenomic replicon and in the enzymatic assays. This resistance was the result of a 250-fold loss in the binding affinity (Kd) of the mutated enzyme to filibuvir. In contrast, the inhibitory activity of VX-222 was only modestly affected by the M423T substitution but more significantly affected by an I482L substitution.

scholarly journals Identification of a C-Terminal Regulatory Motif in Hepatitis C Virus RNA-Dependent RNA Polymerase: Structural and Biochemical Analysis

2003 ◽  
Vol 77 (16) ◽  
pp. 9020-9028 ◽  
Author(s):  
Vincent J.-P. Lévêque ◽  
Robert B. Johnson ◽  
Stephen Parsons ◽  
Jianxin Ren ◽  
Congping Xie ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Dimensional Structure ◽  
Synthesis Rate ◽  
Rna Dependent Rna Polymerase ◽  
Hcv Ns5b ◽  
Ns5b Polymerase

ABSTRACT The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-Å resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to ∼50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.

scholarly journals The C-Terminal Transmembrane Domain of Hepatitis C Virus (HCV) RNA Polymerase Is Essential for HCV Replication In Vivo

2004 ◽  
Vol 78 (7) ◽  
pp. 3797-3802 ◽  
Author(s):  
Ki Jeong Lee ◽  
Jinah Choi ◽  
Jing-hsiung Ou ◽  
Michael M. C. Lai
Keyword(s):  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Transmembrane Domain ◽  
Hcv Rna ◽  
Membrane Association ◽  
Subgenomic Replicon

ABSTRACT Hepatitis C virus (HCV) RNA replication is dependent on the enzymatic activities of the viral RNA-dependent RNA polymerase NS5B, which is a membrane-anchored protein. Recombinant NS5B lacking the C-terminal transmembrane domain (21 amino acids) is enzymatically active. To address the role of this domain in HCV replication in vivo, we introduced a series of mutations into the NS5B of an HCV subgenomic replicon and examined the replication capabilities of the resultant mutants by a colony formation assay. Replicons lacking the transmembrane domain did not yield any colonies. Furthermore, when Huh-7 cells harboring the HCV subgenomic replicon were treated with a synthetic peptide consisting of the NS5B transmembrane domain fused to the antennapedia peptide, the membrane association of NS5B was completely disrupted. Correspondingly, the HCV RNA titer was reduced by approximately 50%. A scrambled peptide used as a control did not have any effects. These findings suggest that the membrane association of NS5B facilitates HCV RNA synthesis. However, a related transmembrane domain derived from bovine viral diarrhea virus could not replace the HCV NS5B transmembrane segment. This finding suggests that the C-terminal 21 amino acids not only have a membrane-anchoring function but also may perform additional functions for RNA synthesis in vivo.

scholarly journals The Mechanism of Action of β-d-2′-Deoxy-2′-Fluoro-2′-C-Methylcytidine Involves a Second Metabolic Pathway Leading to β-d-2′-Deoxy-2′-Fluoro-2′-C-Methyluridine 5′-Triphosphate, a Potent Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

2007 ◽  
Vol 52 (2) ◽  
pp. 458-464 ◽  
Author(s):  
Eisuke Murakami ◽  
Congrong Niu ◽  
Haiying Bao ◽  
Holly M. Micolochick Steuer ◽  
Tony Whitaker ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Potent Inhibitor ◽  
Nucleoside Diphosphate Kinase ◽  
Competitive Inhibitor ◽  
Hcv Rna ◽  
Hepatitis C Virus Rna ◽  
Rna Dependent Rna Polymerase ◽  
Virus Rna

ABSTRACT β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5′-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metabolism of PSI-6130 also results in the formation of the 5′-triphosphate of the uridine congener, β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5′-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, respectively. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymatic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.

scholarly journals Genotypic and Phenotypic Analyses of Hepatitis C Virus Variants Observed in Clinical Studies of VX-222, a Nonnucleoside NS5B Polymerase Inhibitor

2014 ◽  
Vol 58 (9) ◽  
pp. 5456-5465 ◽  
Author(s):  
Min Jiang ◽  
Eileen Z. Zhang ◽  
Andrzej Ardzinski ◽  
Ann Tigges ◽  
Andrew Davis ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Clinical Studies ◽  
Phase 1 ◽  
Replication Capacity ◽  
Hcv Rna ◽  
Genotype 1 ◽  
Wild Type ◽  
Ns5b Polymerase Inhibitor ◽  
Hcv Ns5b

ABSTRACTVX-222, a thiophene-2-carboxylic acid derivative, is a selective nonnucleoside inhibitor of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In phase 1 and 2 clinical studies, VX-222 demonstrated effective antiviral efficacy, with substantial reductions in plasma HCV RNA in patients chronically infected with genotype 1 HCV. To characterize the potential for selection of VX-222-resistant variants in HCV-infected patients, the HCV NS5B gene was sequenced at baseline and during and after 3 days of VX-222 dosing (monotherapy) in a phase 1 study. Variants with the substitutions L419C/I/M/P/S/V, R422K, M423I/T/V, I482L/N/T, A486S/T/V, and V494A were selected during VX-222 dosing, and their levels declined over time after the end of dosing. Phenotypic analysis of these variants was conducted using HCV replicons carrying site-directed mutations. Of the 17 variants, 14 showed reduced susceptibility to VX-222 compared with the wild type, with the L419C/S and R422K variants having higher levels of resistance (>200-fold) than the rest of the variants (6.8- to 76-fold). The M423I and A486S variants remained susceptible to VX-222. The 50% effective concentration (EC50) for the L419P variant could not be obtained due to the poor replication of this replicon. The majority of the variants (15/17) were less fit than the wild type. A subset of the variants, predominately the L419S and R422K variants, were observed when the efficacy and safety of VX-222- and telaprevir-based regimens given for 12 weeks were investigated in genotype 1 HCV-infected patients in a phase 2 study. The NS3 and NS5B variants selected during the dual combination therapy showed reduced susceptibility to both telaprevir and VX-222 and had a lower replication capacity than the wild type. The phase 1b study has the ClinicalTrials.gov identifier NCT00911963, and the phase 2a study has ClinicalTrials.gov identifier NCT01080222.

scholarly journals Regulation of De Novo-Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

2010 ◽  
Vol 84 (12) ◽  
pp. 5923-5935 ◽  
Author(s):  
S. Chinnaswamy ◽  
A. Murali ◽  
P. Li ◽  
K. Fujisaki ◽  
C. C. Kao
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Single Molecule ◽  
Rna Synthesis ◽  
De Novo ◽  
Enzyme Concentration ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the Δ1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the low-resolution conformations of a dimer of a de novo initiation-competent HCV RdRp.

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