scholarly journals Pharmacokinetics of a Cholesterol-conjugated Aptamer Against the Hepatitis C Virus (HCV) NS5B Protein

2015 ◽  
Vol 4 ◽  
pp. e254 ◽  
Author(s):  
Chang Ho Lee ◽  
Soo-Han Lee ◽  
Ji Hyun Kim ◽  
Yook-Hwan Noh ◽  
Gyu-Jeong Noh ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hcv Ns5b ◽  
Ns5b Protein

Allosteric N-acetamide-indole-6-carboxylic acid thumb pocket 1 inhibitors of hepatitis C virus NS5B polymerase — Acylsulfonamides and acylsulfamides as carboxylic acid replacements

2013 ◽  
Vol 91 (1) ◽  
pp. 66-81 ◽  
Author(s):  
Pierre L. Beaulieu ◽  
René Coulombe ◽  
James Gillard ◽  
Christian Brochu ◽  
Jianmin Duan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Carboxylic Acid ◽  
Allosteric Inhibitors ◽  
Subgenomic Replicon ◽  
Structure Activity ◽  
Hcv Ns5b ◽  
Acid Function ◽  
Ns5b Polymerase

Acylsulfonamide and acylsulfamide as surrogates for the carboxylic acid function of N-acetamide-indole-6-carboxylic acids were evaluated as allosteric inhibitors of hepatitis C virus (HCV) NS5B polymerase. Several analogs displayed excellent antiviral potency against both 1a and 1b HCV genotypes in cell-based subgenomic replicon assays. Structure–activity relationships (SAR) are discussed in the context of the crystal structure of an inhibitor − NS5B polymerase complex. Absorption, distribution, metabolism, and excretion pharmacokinetic (ADME-PK) properties of this class of inhibitors are also described.

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 In VitroActivity and Resistance Profile of Dasabuvir, a Nonnucleoside Hepatitis C Virus Polymerase Inhibitor

2014 ◽  
Vol 59 (3) ◽  
pp. 1505-1511 ◽  
Author(s):  
Warren Kati ◽  
Gennadiy Koev ◽  
Michelle Irvin ◽  
Jill Beyer ◽  
Yaya Liu ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Clinical Isolates ◽  
Genotype 1 ◽  
Subgenomic Replicon ◽  
Hcv Genotype ◽  
Genotype 1A ◽  
Treatment Naïve ◽  
Hcv Genotype 1 ◽  
Hcv Ns5b

ABSTRACTDasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.

scholarly journals Ribonucleotide reductase M2 promotes RNA replication of hepatitis C virus by protecting NS5B protein from hPLIC1-dependent proteasomal degradation

2019 ◽  
Vol 294 (15) ◽  
pp. 5759-5773 ◽  
Author(s):  
Bouchra Kitab ◽  
Masaaki Satoh ◽  
Yusuke Ohmori ◽  
Tsubasa Munakata ◽  
Masayuki Sudoh ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Ribonucleotide Reductase ◽  
Rna Replication ◽  
Proteasomal Degradation ◽  
Ns5b Protein

scholarly journals Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

1999 ◽  
Vol 73 (2) ◽  
pp. 1649-1654 ◽  
Author(s):  
Eric Ferrari ◽  
Jacquelyn Wright-Minogue ◽  
Jane W. S. Fang ◽  
Bahige M. Baroudy ◽  
Johnson Y. N. Lau ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Nonstructural Protein ◽  
Full Length ◽  
Dependent Manner ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hydrophobic Domain ◽  
Hcv Ns5b

ABSTRACT Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

scholarly journals Inhibition of Hepatitis C Virus Replication by GS-6620, a PotentC-Nucleoside Monophosphate Prodrug

2014 ◽  
Vol 58 (4) ◽  
pp. 1930-1942 ◽  
Author(s):  
Joy Y. Feng ◽  
Guofeng Cheng ◽  
Jason Perry ◽  
Ona Barauskas ◽  
Yili Xu ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Competitive Inhibitor ◽  
High Dose ◽  
Diarrhea Virus ◽  
A Chain ◽  
Direct Acting ◽  
Hcv Ns5b

ABSTRACTAs a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied thein vitropharmacology of a novelC-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 μM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 μM). The active 5′-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, withKi/Kmvalues of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1′-CN and 2′-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistancein vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with itsin vitroprofile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

scholarly journals Hepatitis C Virus RNA-Dependent RNA Polymerase Is Regulated by Cysteine S-Glutathionylation

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Marina K. Kukhanova ◽  
Vera L. Tunitskaya ◽  
Olga A. Smirnova ◽  
Olga A. Khomich ◽  
Natalia F. Zakirova ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Recombinant Protein ◽  
Rna Polymerase ◽  
Reducing Agents ◽  
Cysteine Residues ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Redox Switches ◽  
Ns5b Protein

Hepatitis C virus (HCV) triggers massive production of reactive oxygen species (ROS) and affects expression of genes encoding ROS-scavenging enzymes. Multiple lines of evidence show that levels of ROS production contribute to the development of various virus-associated pathologies. However, investigation of HCV redox biology so far remained in the paradigm of oxidative stress, whereas no attention was given to the identification of redox switches among viral proteins. Here, we report that one of such redox switches is the NS5B protein that exhibits RNA-dependent RNA polymerase (RdRp) activity. Treatment of the recombinant protein with reducing agents significantly increases its enzymatic activity. Moreover, we show that the NS5B protein is subjected to S-glutathionylation that affects cysteine residues 89, 140, 170, 223, 274, 521, and either 279 or 295. Substitution of these cysteines except C89 and C223 with serine residues led to the reduction of the RdRp activity of the recombinant protein in a primer-dependent assay. The recombinant protein with a C279S mutation was almost inactive in vitro and could not be activated with reducing agents. In contrast, cysteine substitutions in the NS5B region in the context of a subgenomic replicon displayed opposite effects: most of the mutations enhanced HCV replication. This difference may be explained by the deleterious effect of oxidation of NS5B cysteine residues in liver cells and by the protective role of S-glutathionylation. Based on these data, redox-sensitive posttranslational modifications of HCV NS5B and other proteins merit a more detailed investigation and analysis of their role(s) in the virus life cycle and associated pathogenesis.

scholarly journals Quasispecies dynamics in hepatitis C liver transplant recipients receiving grafts from hepatitis C virus infected donors

2015 ◽  
Vol 96 (12) ◽  
pp. 3493-3498 ◽  
Author(s):  
Sofía Pérez-del-Pulgar ◽  
Josep Gregori ◽  
Francisco Rodríguez-Frías ◽  
Patricia González ◽  
Damir García-Cehic ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Liver Transplant ◽  
Viral Population ◽  
Sequencing Analysis ◽  
Homogeneous Population ◽  
Infected Liver ◽  
The One ◽  
Hcv Ns5b ◽  
Liver Transplant Recipients

The allocation of liver grafts from hepatitis C virus (HCV)-positive donors in HCV-infected liver transplant (LT) recipients leads to infection with two different viral populations. In a previous study, we examined quasispecies dynamics during reinfection by clonal sequencing, which did not allow an accurate characterization of coexistence and competition events. To overcome this limitation, here we used deep-sequencing analysis of a fragment of the HCV NS5B gene in six HCV-infected LT recipients who received HCV-infected grafts. Successive expansions and contractions of quasispecies complexity were observed, evolving in all cases towards a more homogeneous population. The population that became dominant was the one displaying the highest mutant spectrum complexity. In four patients, coexistence of minority mutants, derived from the donor or the recipient, were detected. In conclusion, our study shows that, during reinfection with a different HCV strain in LT recipients, the viral population with the highest diversity always becomes dominant.

scholarly journals Effect of Hepatitis C Virus (HCV) NS5B-Nucleolin Interaction on HCV Replication with HCV Subgenomic Replicon

2006 ◽  
Vol 80 (7) ◽  
pp. 3332-3340 ◽  
Author(s):  
Tetsuro Shimakami ◽  
Masao Honda ◽  
Takashi Kusakawa ◽  
Takayuki Murata ◽  
Kunitada Shimotohno ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Polymerase Activity ◽  
Subgenomic Replicon ◽  
Huh7 Cells ◽  
Hcv Ns5b

ABSTRACT We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

scholarly journals De Novo Initiation of RNA Synthesis by Hepatitis C Virus Nonstructural Protein 5B Polymerase

2000 ◽  
Vol 74 (4) ◽  
pp. 2017-2022 ◽  
Author(s):  
Weidong Zhong ◽  
Annette S. Uss ◽  
Eric Ferrari ◽  
Johnson Y. N. Lau ◽  
Zhi Hong
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
De Novo ◽  
Rna Viruses ◽  
Rna Replication ◽  
Nucleoside Triphosphate ◽  
Positive Strand ◽  
Positive Strand Rna ◽  
Hcv Ns5b

ABSTRACT RNA-dependent RNA polymerase (RdRp) encoded by positive-strand RNA viruses is critical to the replication of viral RNA genome. Like other positive-strand RNA viruses, replication of hepatitis C virus (HCV) RNA is mediated through a negative-strand intermediate, which is generated through copying the positive-strand genomic RNA. Although it has been demonstrated that HCV NS5B alone can direct RNA replication through a copy-back primer at the 3′ end, de novo initiation of RNA synthesis is likely to be the mode of RNA replication in infected cells. In this study, we demonstrate that a recombinant HCV NS5B protein has the ability to initiate de novo RNA synthesis in vitro. The NS5B used HCV 3′ X-tail RNA (98 nucleotides) as the template to synthesize an RNA product of monomer size, which can be labeled by [γ-32P]nucleoside triphosphate. The de novo initiation activity was further confirmed by using small synthetic RNAs ending with dideoxynucleotides at the 3′ termini. In addition, HCV NS5B preferred GTP as the initiation nucleotide. The optimal conditions for the de novo initiation activity have been determined. Identification and characterization of the de novo priming or initiation activity by HCV NS5B provides an opportunity to screen for inhibitors that specifically target the initiation step.

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