scholarly journals Interaction with a Ubiquitin-Like Protein Enhances the Ubiquitination and Degradation of Hepatitis C Virus RNA-Dependent RNA Polymerase

2003 ◽  
Vol 77 (7) ◽  
pp. 4149-4159 ◽  
Author(s):  
Lu Gao ◽  
Hong Tu ◽  
Stephanie T. Shi ◽  
Ki-Jeong Lee ◽  
Miyuki Asanaka ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase ◽  
Subgenomic Replicon ◽  
C Terminus ◽  
Huh7 Cells

ABSTRACT To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.

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 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 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.

Potential Inhibition of COVID-19 RNA-dependent RNA Polymerase by Hepatitis C Virus Non-nucleoside Inhibitors: An In-silico Perspective

2020 ◽  
Vol 17 ◽  
Author(s):  
Yee Siew Choong ◽  
Theam Soon Lim ◽  
Hanyun Liu ◽  
Rubin Jiang ◽  
Zimu Cai ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Binding Sites ◽  
Homology Modelling ◽  
Clinical Importance ◽  
Rna Dependent Rna Polymerase ◽  
Nucleoside Inhibitors

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a novel member of the genus betacoronavirus in the Coronaviridae family. It has been identified as the causative agent of coronavirus disease 2019 (COVID-19) spreading rapidly in Asia, America and Europe. Like some other RNA viruses, RNA replication and transcription of SARS-CoV-2 relies on its RNA-dependent RNA polymerase (RdRP), which is a therapeutic target of clinical importance. Crystal structure of SARS-CoV-2 that was solved recently (PDB ID 6M71) with some missing residues. Objective: We used SARS-CoV-2 RdRP as a target protein to screen for possible chemical molecules with potential antiviral effects. Method: Here we modelled the missing residues 896-905 via homology modelling and then analysed the interactions of Hepatitis C virus allosteric non-nucleoside inhibitors (NNIs) in the reported NNIs binding sites in SARS-CoV-2 RdRP. Results and Discussion: We found that MK-3281, filibuvir, setrobuvir and dasabuvir might be able to inhibit SARS-CoV-2 RdRP based on their binding affinities in the respective binding sites. Conclusion: Further in vitro and in vivo experimental research will be carried out to evaluate their effectiveness in COVID19 treatment in the near future.

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.

Monitoring hepatitis C virus (HCV) RNA-dependent RNA polymerase oligomerization by a FRET-based in vitro system

2010 ◽  
Vol 87 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Itxaso Bellón-Echeverría ◽  
Alberto José López-Jiménez ◽  
Pilar Clemente-Casares ◽  
Antonio Mas
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase ◽  
In Vitro System

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.

THE QSAR RESEARCH ON AURONE INHIBITORS OF HEPATITIS C VIRUS RNA-DEPENDENT RNA POLYMERASE BY APPLYING MULTI-DIMENSIONAL SCALING METHOD

2013 ◽  
Vol 06 (01) ◽  
pp. 1250062
Author(s):  
YONG-HONG HU ◽  
BAO-HUA ZHANG
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Hcv Rna ◽  
Training Set ◽  
Rna Dependent Rna Polymerase ◽  
Dimensional Scaling ◽  
Naturally Occurring ◽  
Virus Rna ◽  
Multi Dimensional Scaling

In this paper, we take naturally occurring 2-benzylidenebenzofuran-3-ones (aurones) inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) as an example to study the Multi-dimensional scaling (MDS) method for structure-activity relationship. By analyzing training set molecules, our MDS method combined with a PROXSCAL algorithm can predict inhibitory activity of most compounds correctly. Thus, a new sample's activity can be estimated and judged conveniently, and whether it should be synthesized can be known. The MDS method is applicable to optimize the structure for a compound and to provide suggestions for drug design.

scholarly journals A role for B cells to transmit hepatitis C virus infection

2020 ◽  
Author(s):  
Isabelle Desombere ◽  
Freya Van Houtte ◽  
Ali Farhoudi ◽  
Lieven Verhoye ◽  
Caroline Buysschaert ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
B Cells ◽  
Neutralizing Antibody ◽  
Hcv Rna ◽  
Serum Samples ◽  
Cell Culture Systems ◽  
Hcv Transmission

Abstract Hepatitis C virus (HCV) is highly variable and transmits through infected blood to establish a chronic liver infection in the majority of patients. Our knowledge of the infectivity of clinical HCV strains is hampered by the lack of in vitro cell culture systems that support efficient viral replication. We previously reported that laboratory strains of HCV associated with non-permissive B cells could trans-infect hepatocytes and thereby evade host neutralizing antibody responses, suggesting a role for B cells in HCV transmission. To evaluate this hypothesis, we assessed the ability of B cells and sera from recent (<2 years) or chronic (≥ 2 years) infections to infect humanized liver chimeric mice. HCV was efficiently transmitted by B cells from chronically infected patients whereas the sera were non-infectious. In contrast, we noted that B cells from recently infected patients failed to transmit HCV to the mice, whereas all serum samples were infectious. Only patients with circulating anti-glycoprotein antibodies harbored genomic HCV-RNA in B cells. Taken together, our studies provide direct in vivo evidence for HCV transmission by B cells and these findings may have clinical implications for prophylactic and therapeutic antibody-based vaccine design.

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