scholarly journals A Fluorescence-Based High-Throughput Screen to Identify Small Compound Inhibitors of the Genotype 3a Hepatitis C Virus RNA Polymerase

2013 ◽  
Vol 18 (9) ◽  
pp. 1027-1034 ◽  
Author(s):  
Auda A. Eltahla ◽  
Kurt Lackovic ◽  
Christopher Marquis ◽  
John-Sebastian Eden ◽  
Peter A. White
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
High Throughput ◽  
High Throughput Screening ◽  
De Novo ◽  
Specific Activity ◽  
Hcv Rna ◽  
High Throughput Screen ◽  
Rdrp Activity

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) plays an essential role in the replication of HCV and is a key target for novel antiviral therapies. Several RdRp inhibitors are in clinical trials and have increased response rates when combined with current interferon-based therapies for genotype 1 (G1) HCV patients. These inhibitors, however, show poor efficacy against non-G1 genotypes, including G3a, which represents ~20% of HCV cases globally. Here, we used a commercially available fluorescent dye to characterize G3a HCV RdRp in vitro. RdRp activity was assessed via synthesis of double-stranded RNA from the single-stranded RNA poly(C) template. The assay was miniaturized to a 384-well microplate format and a pilot high-throughput screen was conducted using 10,208 “lead-like” compounds, randomly selected to identify inhibitors of HCV G3a RdRp. Of 150 compounds demonstrating greatest inhibition, 10 were confirmed using both fluorescent and radioactive assays. The top two inhibitors (HAC001 and HAC002) demonstrated specific activity, with an IC50 of 12.7 µM and 1.0 µM, respectively. In conclusion, we describe simple, fluorescent-based high-throughput screening (HTS) for the identification of inhibitors of de novo RdRp activity, using HCV G3a RdRp as the target. The HTS system could be used against any positive-sense RNA virus that cannot be cultured.

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 Hydrophobic and Charged Residues in the C-Terminal Arm of Hepatitis C Virus RNA-Dependent RNA Polymerase Regulate Initiation and Elongation

2014 ◽  
Vol 89 (4) ◽  
pp. 2052-2063 ◽  
Author(s):  
Amy L. Cherry ◽  
Caitriona A. Dennis ◽  
Andrew Baron ◽  
Leslie E. Eisele ◽  
Pia A. Thommes ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
De Novo ◽  
Structural Features ◽  
Primer Extension ◽  
Hcv Rna ◽  
Genome Replication ◽  
Rna Dependent Rna Polymerase ◽  
Subgenomic Replicon

ABSTRACTThe RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a β-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of β-loop and C-terminal arm mutants, which were used forin vitroanalysis of RdRpde novoinitiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the β-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general.IMPORTANCEHCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr→Phe or Asp→Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

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 Structural Analysis of the Hepatitis C Virus RNA Polymerase in Complex with Ribonucleotides

2002 ◽  
Vol 76 (7) ◽  
pp. 3482-3492 ◽  
Author(s):  
Stéphane Bressanelli ◽  
Licia Tomei ◽  
Félix A. Rey ◽  
Raffaele De Francesco
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
De Novo ◽  
Crystallographic Analysis ◽  
Catalytic Site ◽  
Molecular Surface ◽  
Hcv Rna ◽  
Divalent Metal Ions ◽  
Virus Rna

ABSTRACT We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 Å away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The electron density map at 1.7-Å resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage φ6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the φ6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.

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 De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

2004 ◽  
Vol 78 (22) ◽  
pp. 12207-12217 ◽  
Author(s):  
C. T. Ranjith-Kumar ◽  
R. T. Sarisky ◽  
L. Gutshall ◽  
M. Thomson ◽  
C. C. Kao
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
De Novo ◽  
Specific Interaction ◽  
Binding Pocket ◽  
Hcv Rna ◽  
Elongation Complex ◽  
Rna Dependent Rna Polymerase ◽  
Template Switch

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a Km for GTP of 3.5 μM, all of the mutations except one had Km s that were three- to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.

scholarly journals Novel Nonnucleoside Inhibitor of Hepatitis C Virus RNA-Dependent RNA Polymerase

2004 ◽  
Vol 48 (12) ◽  
pp. 4813-4821 ◽  
Author(s):  
Anita Y. M. Howe ◽  
Johnathan Bloom ◽  
Carl J. Baldick ◽  
Christopher A. Benetatos ◽  
Huiming Cheng ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
High Throughput Screening ◽  
Viral Rna ◽  
Alpha Interferon ◽  
Hcv Rna ◽  
Binding Studies ◽  
Rna Dependent Rna Polymerase ◽  
Multiple Treatments

ABSTRACT A novel nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), [(1R)-5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyano[3,4-b]indol-1-yl] acetic acid (HCV-371), was discovered through high-throughput screening followed by chemical optimization. HCV-371 displayed broad inhibitory activities against the NS5B RdRp enzyme, with 50% inhibitory concentrations ranging from 0.3 to 1.8 μM for 90% of the isolates derived from HCV genotypes 1a, 1b, and 3a. HCV-371 showed no inhibitory activity against a panel of human polymerases, including mitochondrial DNA polymerase gamma, and other unrelated viral polymerases, demonstrating its specificity for the HCV polymerase. A single administration of HCV-371 to cells containing the HCV subgenomic replicon for 3 days resulted in a dose-dependent reduction of the steady-state levels of viral RNA and protein. Multiple treatments with HCV-371 for 16 days led to a >3-log10 reduction in the HCV RNA level. In comparison, multiple treatments with a similar inhibitory dose of alpha interferon resulted in a 2-log10 reduction of the viral RNA level. In addition, treatment of cells with a combination of HCV-371 and pegylated alpha interferon resulted in an additive antiviral activity. Within the effective antiviral concentrations of HCV-371, there was no effect on cell viability and metabolism. The intracellular antiviral specificity of HCV-371 was demonstrated by its lack of activity in cells infected with several DNA or RNA viruses. Fluorescence binding studies show that HCV-371 binds the NS5B with an apparent dissociation constant of 150 nM, leading to high selectivity and lack of cytotoxicity in the antiviral assays.

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.

scholarly journals Dinucleotide Analogues as Novel Inhibitors of RNA-Dependent RNA Polymerase of Hepatitis C Virus

2003 ◽  
Vol 47 (8) ◽  
pp. 2674-2681 ◽  
Author(s):  
Weidong Zhong ◽  
Haoyun An ◽  
Dinesh Barawkar ◽  
Zhi Hong
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Replication Initiation ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase ◽  
Initiation Process

ABSTRACT Replication of hepatitis C virus (HCV) RNA is catalyzed by the virally encoded RNA-dependent RNA polymerase NS5B. It is believed that the viral polymerase utilizes a de novo or primer-independent mechanism for initiation of RNA synthesis. Our previous work has shown that dinucleotides were efficient initiation molecules for NS5B in vitro (W. Zhong, E. Ferrari, C. A. Lesburg, D. Maag, S. K. Ghosh, C. E. Cameron, J. Y. Lau, and Z. Hong, J. Virol. 74:9134-9143, 2000). In this study, we further demonstrated that dinucleotide analogues could serve as inhibitors of de novo initiation of RNA synthesis directed by HCV NS5B. Both mononucleotide- and dinucleotide-initiated RNA syntheses were affected by dinucleotide analogues. The presence of the 5′-phosphate group in the dinucleotide compounds was required for efficient inhibition of de novo initiation. Optimal inhibitory activity also appeared to be dependent on the base-pairing potential between the compounds and the template terminal bases. Because the initiation process is a rate-limiting step in viral RNA replication, inhibitors that interfere with the initiation process will have advantages in suppressing virus replication. The use of dinucleotide analogues as inhibitor molecules to target viral replication initiation represents a novel approach to antiviral interference.

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