scholarly journals Characterization of Hepatitis C Virus Subgenomic Replicon Resistance to Cyclosporine In Vitro

2007 ◽  
Vol 81 (11) ◽  
pp. 5829-5840 ◽  
Author(s):  
John M. Robida ◽  
Heather B. Nelson ◽  
Zhe Liu ◽  
Hengli Tang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cellular Protein ◽  
Live Cells ◽  
Hcv Rna ◽  
Coding Region ◽  
Subgenomic Replicon ◽  
Cyclophilin B

ABSTRACT Treatment of hepatitis C virus (HCV) infection has been met with less than satisfactory results due primarily to its resistance to and significant side effects from alpha interferon (IFN-α). New classes of safe and broadly acting treatments are urgently needed. Cyclosporine (CsA), an immunosuppressive and anti-inflammatory drug for organ transplant patients, has recently been shown to be highly effective in suppressing HCV replication through a mechanism that is distinct from the IFN pathway. Here we report the selection and characterization of HCV replicon cells that are resistant to CsA treatment in vitro, taking advantage of our ability to sort live cells that are actively replicating HCV RNA in the presence of drug treatments. This resistance is specific to CsA as the replicon cells most resistant to CsA were still sensitive to IFN-α and a polymerase inhibitor. We demonstrate that the resistant phenotype is not a result of general enhanced replication and, furthermore, that mutations in the coding region of HCV NS5B contribute to the resistance. Interestingly, a point mutation (I432V) isolated from the most resistant replicon was able to rescue a lethal mutation (P540A) in NS5B that disrupts its interaction with its cofactor, cyclophilin B (CypB), even though the I432V mutation is located outside of the reported CypB binding site (amino acids 520 to 591). Our results demonstrate that CsA exerts selective pressure on the HCV genome, leading to the emergence of resistance-conferring mutations in the viral genome despite acting upon a cellular protein.

scholarly journals Genomic-Scale Interaction Involving Complementary Sequences in the Hepatitis C Virus 5′UTR Domain IIa and the RNA-Dependent RNA Polymerase Coding Region Promotes Efficient Virus Replication

Viruses ◽  
2018 ◽  
Vol 11 (1) ◽  
pp. 17 ◽  
Author(s):  
Elodie Rance ◽  
Jerome Tanner ◽  
Caroline Alfieri
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Progeny Virus ◽  
Hcv Rna ◽  
Genomic Rna ◽  
Coding Region ◽  
Rna Dependent Rna Polymerase ◽  
High Affinity

The hepatitis C virus (HCV) genome contains structured elements thought to play important regulatory roles in viral RNA translation and replication processes. We used in vitro RNA binding assays to map interactions involving the HCV 5′UTR and distal sequences in NS5B to examine their impact on viral RNA replication. The data revealed that 5′UTR nucleotides (nt) 95–110 in the internal ribosome entry site (IRES) domain IIa and matching nt sequence 8528–8543 located in the RNA-dependent RNA polymerase coding region NS5B, form a high-affinity RNA-RNA complex in vitro. This duplex is composed of both wobble and Watson-Crick base-pairings, with the latter shown to be essential to the formation of the high-affinity duplex. HCV genomic RNA constructs containing mutations in domain IIa nt 95–110 or within the genomic RNA location comprising nt 8528–8543 displayed, on average, 5-fold less intracellular HCV RNA and 6-fold less infectious progeny virus. HCV genomic constructs containing complementary mutations for IRES domain IIa nt 95–110 and NS5B nt 8528–8543 restored intracellular HCV RNA and progeny virus titers to levels obtained for parental virus RNA. We conclude that this long-range duplex interaction between the IRES domain IIa and NS5B nt 8528–8543 is essential for optimal virus replication.

scholarly journals cis-Acting RNA Signals in the NS5B C-Terminal Coding Sequence of the Hepatitis C Virus Genome

2004 ◽  
Vol 78 (20) ◽  
pp. 10865-10877 ◽  
Author(s):  
Haekyung Lee ◽  
Hyukwoo Shin ◽  
Eckard Wimmer ◽  
Aniko V. Paul
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Genome ◽  
Coding Region ◽  
Subgenomic Replicon ◽  
Stem Loop ◽  
Coding Sequence ◽  
Cis Acting ◽  
Rna Elements

ABSTRACT The cis-replicating RNA elements in the 5′ and 3′ nontranslated regions (NTRs) of the hepatitis C virus (HCV) genome have been thoroughly studied before. However, no cis-replicating elements have been identified in the coding sequences of the HCV polyprotein until very recently. The existence of highly conserved and stable stem-loop structures in the RNA polymerase NS5B coding sequence, however, has been previously predicted (A. Tuplin, J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds, RNA 8:824-841, 2002). We have selected for our studies a 249-nt-long RNA segment in the C-terminal NS5B coding region (NS5BCR), which is predicted to form four stable stem-loop structures (SL-IV to SL-VII). By deletion and mutational analyses of the RNA structures, we have determined that two of the stem-loops (SL-V and SL-VI) are essential for replication of the HCV subgenomic replicon in Huh-7 cells. Mutations in the loop and the top of the stem of these RNA elements abolished replicon RNA synthesis but had no effect on translation. In vitro gel shift and filter-binding assays revealed that purified NS5B specifically binds to SL-V. The NS5B-RNA complexes were specifically competed away by unlabeled homologous RNA, to a small extent by 3′ NTR RNA, and only poorly by 5′ NTR RNA. The other two stem-loops (SL-IV and SL-VII) of the NS5BCR domain were found to be important but not essential for colony formation by the subgenomic replicon. The precise function(s) of these cis-acting RNA elements is not known.

scholarly journals In Vitro RNA Replication Directed by Replicase Complexes Isolated from the Subgenomic Replicon Cells of Hepatitis C Virus

2003 ◽  
Vol 77 (3) ◽  
pp. 2295-2300 ◽  
Author(s):  
Vicky C. H. Lai ◽  
Shannon Dempsey ◽  
Johnson Y. N. Lau ◽  
Zhi Hong ◽  
Weidong Zhong
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
Divalent Cations ◽  
Hcv Rna ◽  
Nonstructural Proteins ◽  
Subgenomic Replicon ◽  
Free System ◽  
Potential Inhibitors

ABSTRACT Replication of hepatitis C virus (HCV) RNA in virus-infected cells is believed to be catalyzed by viral replicase complexes (RCs), which may consist of various virally encoded nonstructural proteins and host factors. In this study, we characterized the RC activity of a crude membrane fraction isolated from HCV subgenomic replicon cells. The RC preparation was able to use endogenous replicon RNA as a template to synthesize both single-stranded (ss) and double-stranded (ds) RNA products. Divalent cations (Mg2+ and Mn2+) showed different effects on RNA synthesis. Mg2+ ions stimulated the synthesis of ss RNA but had little effect on the synthesis of ds RNA. In contrast, Mn2+ ions enhanced primarily the synthesis of ds RNA. Interestingly, ss RNA could be synthesized under certain conditions in the absence of ds RNA, and vice versa, suggesting that the ss and ds RNA were derived either from different forms of replicative intermediates or from different RCs. Pulse-chase analysis showed that radioactivity incorporated into the ss RNA was chased into the ds RNA and other larger RNA species. This observation indicated that the newly synthesized ss RNA could serve as a template for a further round of RNA synthesis. Finally, 3′ deoxyribonucleoside triphosphates were able to inhibit RNA synthesis in this cell-free system, presumably through chain termination, with 3′ dGTP having the highest potency. Establishment of the replicase assay will facilitate the identification and evaluation of potential inhibitors that would act against the entire RC of HCV.

scholarly journals VX-950, a Novel Hepatitis C Virus (HCV) NS3-4A Protease Inhibitor, Exhibits Potent Antiviral Activities in HCV Replicon Cells

2006 ◽  
Vol 50 (5) ◽  
pp. 1813-1822 ◽  
Author(s):  
Kai Lin ◽  
Robert B. Perni ◽  
Ann D. Kwong ◽  
Chao Lin
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Agents ◽  
Alpha Interferon ◽  
Hcv Rna ◽  
Resistance Mutations ◽  
In Vitro Characterization ◽  
Antiviral Activities

ABSTRACT The NS3-4A serine protease of hepatitis C virus (HCV) is essential for viral replication and therefore has been one of the most attractive targets for developing specific antiviral agents against HCV. VX-950, a highly selective, reversible, and potent peptidomimetic inhibitor of the HCV NS3-4A protease, is currently in clinical development for the treatment of hepatitis C. In this report, we describe the in vitro characterization of anti-HCV activities of VX-950 in subgenomic HCV replicon cells. Incubation with VX-950 resulted in a time- and dose-dependent reduction of HCV RNA and proteins in replicon cells. Moreover, following a 2-week incubation with VX-950, a reduction in HCV RNA levels of 4.7 log10 was observed, and this reduction resulted in elimination of HCV RNA from replicon cells, since there was no rebound in replicon RNA after withdrawal of the inhibitor. The combination of VX-950 and alpha interferon was additive to moderately synergistic in reducing HCV RNA in replicon cells with no significant increase in cytotoxicity. The benefit of the combination was sustained over time: a 4-log10 reduction in HCV RNA level was achieved following a 9-day incubation with VX-950 and alpha interferon at lower concentrations than when either VX-950 or alpha interferon was used alone. The combination of VX-950 and alpha interferon also suppressed the emergence of in vitro resistance mutations against VX-950 in replicon cells.

scholarly journals Hepatitis C Virus Genotype 5a Subgenomic Replicons for Evaluation of Direct-Acting Antiviral Agents

2014 ◽  
Vol 58 (9) ◽  
pp. 5386-5394 ◽  
Author(s):  
Constance N. Wose Kinge ◽  
Christine Espiritu ◽  
Nishi Prabdial-Sing ◽  
Nomathamsaqa Patricia Sithebe ◽  
Mohsan Saeed ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Antiviral Agents ◽  
Rna Replication ◽  
Firefly Luciferase ◽  
Hcv Rna ◽  
Virus Genotype ◽  
Subgenomic Replicon

ABSTRACTHepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy.In vitroreplication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established anin vitroreplication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds.

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 Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase

2020 ◽  
Vol 295 (35) ◽  
pp. 12426-12436 ◽  
Author(s):  
Lorillee Tallorin ◽  
Valerie A. Villareal ◽  
Chih-Yun Hsia ◽  
Mary A. Rodgers ◽  
Dominique J. Burri ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Lipid Metabolism ◽  
Hepatitis C ◽  
Molecular Mechanisms ◽  
Rna Replication ◽  
Hcv Rna ◽  
Genome Replication ◽  
Subgenomic Replicon ◽  
Lipid Environment

Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

scholarly journals Mobility analysis of an NS5A–GFP fusion protein in cells actively replicating hepatitis C virus subgenomic RNA

2007 ◽  
Vol 88 (2) ◽  
pp. 470-475 ◽  
Author(s):  
Daniel M. Jones ◽  
Sarah N. Gretton ◽  
John McLauchlan ◽  
Paul Targett-Adams
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fluorescent Proteins ◽  
Viral Proteins ◽  
Hcv Rna ◽  
Coding Region ◽  
Subgenomic Replicon ◽  
Subgenomic Rna ◽  
Photoactivatable Gfp ◽  
In Cells

We have introduced GFP and photoactivatable GFP into the NS5A coding region of a hepatitis C virus (HCV) subgenomic replicon that gives efficient transient replication. NS5A–GFP, expressed by the replicon, could be detected in cytoplasmic fluorescent foci as early as 4 h after RNA was introduced into cells. The fluorescent foci are likely to be sites where RNA synthesis could occur, although their production was not dependent on prior replication. Photobleaching studies demonstrated that the fluorescent proteins were relatively immobile upon expression from replicon RNAs. By contrast, an NS5A–GFP chimera produced in the absence of other viral proteins was mobile. Hence, interactions in cells expressing HCV replication proteins limit NS5A mobility, and transfer of viral proteins between foci is either slow or does not occur. Thus, the sites of HCV RNA replication possibly have a fixed complement of proteins that may act as discrete factories for producing viral RNA.

scholarly journals Genotypic and Phenotypic Analyses of Hepatitis C Virus from Patients Treated with JTK-853 in a Three-Day Monotherapy

2012 ◽  
Vol 57 (1) ◽  
pp. 436-444 ◽  
Author(s):  
Naoki Ogura ◽  
Yukiyo Toyonaga ◽  
Izuru Ando ◽  
Kunihiro Hirahara ◽  
Tsutomu Shibata ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Viral Resistance ◽  
Hcv Rna ◽  
Amino Acid Substitutions ◽  
Sequencing Analysis ◽  
Resistance Mutations ◽  
In The Wild

ABSTRACTJTK-853, a palm site-binding NS5B nonnucleoside polymerase inhibitor, shows antiviral activityin vitroand in hepatitis C virus (HCV)-infected patients. Here, we report the results of genotypic and phenotypic analyses of resistant variants in 24 HCV genotype 1-infected patients who received JTK-853 (800, 1,200, or 1,600 mg twice daily or 1,200 mg three times daily) in a 3-day monotherapy. Viral resistance in NS5B was investigated using HCV RNA isolated from serum specimens from the patients. At the end of treatment (EOT) with JTK-853, the amino acid substitutions M414T (methionine [M] in position 414 at baseline was replaced with threonine [T] at EOT), C445R (cysteine [C] in position 445 at baseline was replaced with arginine [R] at EOT), Y448C/H (tyrosine [Y] in position 448 at baseline was replaced with cysteine [C] or histidine [H] at EOT), and L466F (leucine [L] in position 466 at baseline was replaced with phenylalanine [F] at EOT), which are known to be typical resistant variants of nonnucleoside polymerase inhibitors, were observed in a clonal sequencing analysis. These substitutions were also selected by a treatment with JTK-853in vitro, and the 50% effective concentration of JTK-853 in the M414T-, C445F-, Y448H-, and L466V-harboring replicons attenuated the susceptibility by 44-, 5-, 6-, and 21-fold, respectively, compared with that in the wild-type replicon (Con1). These findings suggest that amino acid substitutions of M414T, C445R, Y448C/H, and L466F are thought to be viral resistance mutations in HCV-infected patients receiving JTK-853 in a 3-day monotherapy.

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