scholarly journals Interleukin-1 Inhibits Hepatitis C Virus Subgenomic RNA Replication by Activation of Extracellular Regulated Kinase Pathway

2003 ◽  
Vol 77 (9) ◽  
pp. 5493-5498 ◽  
Author(s):  
Haizhen Zhu ◽  
Chen Liu
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Interleukin 1 ◽  
Rna Replication ◽  
Inhibitory Effect ◽  
Chronic Infections ◽  
Subgenomic Replicon ◽  
Subgenomic Rna ◽  
Interferon Stimulated Genes

ABSTRACT Interleukin-1 (IL-1) plays an important role in the inflammatory process. Some studies have demonstrated that IL-1 production was impaired in patients with chronic infections of hepatitis C virus (HCV), implying that IL-1 may play a role in viral clearance. Using an HCV subgenomic replicon cell line, we demonstrate that IL-1 can effectively inhibit HCV subgenomic RNA replication and viral protein expression, suggesting that IL-1 has direct antiviral activity. The inhibitory effect is associated with the extracellular regulatory kinase (ERK) activation. In addition, we also show that IL-1 can induce one of the interferon-stimulated genes (ISGs), 1-8U, which exhibits antiviral activity. However, it has no effect on the other ISG, 6-16, suggesting that IL-1 induces novel antiviral pathways within a cell.

scholarly journals Development of Intergenotypic Chimeric Replicons To Determine the Broad-Spectrum Antiviral Activities of Hepatitis C Virus Polymerase Inhibitors

2008 ◽  
Vol 52 (10) ◽  
pp. 3523-3531 ◽  
Author(s):  
Koleen J. Herlihy ◽  
Joanne P. Graham ◽  
Robert Kumpf ◽  
Amy K. Patick ◽  
Rohit Duggal ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Broad Spectrum ◽  
Inhibitor Binding ◽  
Subgenomic Replicon ◽  
Activity Determination ◽  
Polymerase Inhibitors ◽  
Overlap Extension

ABSTRACT To address the need for broad-spectrum antiviral activity characterization of hepatitis C virus (HCV) polymerase inhibitors, we created a panel of intergenotypic chimeric replicons containing nonstructural (NS) protein NS5B sequences from genotype 2b (GT2b), GT3a, GT4a, GT5a, and GT6a HCV isolates. Viral RNA extracted from non-GT1 HCV patient plasma was subjected to reverse transcription. The NS5B region was amplified by nested PCR and introduced into the corresponding region of the GT1b (Con-1) subgenomic reporter replicon by Splicing by Overlap Extension (SOEing) PCR. Stable cell lines were generated with replication-competent chimeras for in vitro antiviral activity determination of HCV nonnucleoside polymerase inhibitors (NNIs) that target different regions of the protein. Compounds that bind to the NNI2 (thiophene carboxylic acid) or NNI3 (benzothiadiazine) allosteric sites showed 8- to >1,280-fold reductions in antiviral activity against non-GT1 NS5B chimeric replicons compared to that against the GT1b subgenomic replicon. Smaller reductions in susceptibility, ranging from 0.2- to 33-fold, were observed for the inhibitor binding to the NNI1 (benzimidazole) site. The inhibitor binding to the NNI4 (benzofuran) site showed broad-spectrum antiviral activity against all chimeric replicons evaluated in this study. In conclusion, evaluation of HCV NNIs against intergenotypic chimeric replicons showed differences in activity spectrum for inhibitors that target different regions of the enzyme, some of which could be associated with specific residues that differ between GT1 and non-GT1 polymerases. Our study demonstrates the utility of chimeric replicons for broad-spectrum activity determination of HCV inhibitors.

scholarly journals Palmitoylation and Polymerization of Hepatitis C Virus NS4B Protein

2006 ◽  
Vol 80 (12) ◽  
pp. 6013-6023 ◽  
Author(s):  
Guann-Yi Yu ◽  
Ki-Jeong Lee ◽  
Lu Gao ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Membrane Structure ◽  
Rna Replication ◽  
Polymerization Process ◽  
Cysteine Residues ◽  
Hcv Rna ◽  
Subgenomic Replicon ◽  
Lipid Modifications ◽  
Ns4b Protein

ABSTRACT Hepatitis C Virus (HCV) NS4B protein induces a specialized membrane structure which may serve as the replication platform for HCV RNA replication. In the present study, we demonstrated that NS4B has lipid modifications (palmitoylation) on two cysteine residues (cysteines 257 and 261) at the C-terminal end. Site-specific mutagenesis of these cysteine residues on individual NS4B proteins and on an HCV subgenomic replicon showed that the lipid modifications, particularly of Cys261, are important for protein-protein interaction in the formation of the HCV RNA replication complex. We further demonstrated that NS4B can undergo polymerization. The main polymerization determinants were mapped in the N-terminal cytosolic domain of NS4B protein; however, the lipid modifications on the C terminus also facilitate the polymerization process. The lipid modification and the polymerization activity could be two properties of NS4B important for its induction of the specialized membrane structure involved in viral RNA replication.

scholarly journals Mutations in NS5B Polymerase of Hepatitis C Virus: Impacts on in Vitro Enzymatic Activity and Viral RNA Replication in the Subgenomic Replicon Cell Culture

Virology ◽  
2002 ◽  
Vol 297 (2) ◽  
pp. 298-306 ◽  
Author(s):  
I.Wayne Cheney ◽  
Suhaila Naim ◽  
Vicky C.H. Lai ◽  
Shannon Dempsey ◽  
Daniel Bellows ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Enzymatic Activity ◽  
Rna Replication ◽  
Viral Rna ◽  
Subgenomic Replicon ◽  
Replicon Cell ◽  
Ns5b Polymerase

scholarly journals Dehydrojuncusol, a Natural Phenanthrene Compound Extracted fromJuncus maritimus, Is a New Inhibitor of Hepatitis C Virus RNA Replication

2019 ◽  
Vol 93 (10) ◽  
Author(s):  
Marie-Emmanuelle Sahuc ◽  
Ramla Sahli ◽  
Céline Rivière ◽  
Véronique Pène ◽  
Muriel Lavie ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Treatment Failure ◽  
Natural Compound ◽  
Rna Replication ◽  
Direct Acting Antivirals ◽  
Ns5a Protein ◽  
Juncus Maritimus ◽  
Direct Acting

ABSTRACTRecent emergence of direct-acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA-resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, the search for new inhibitors with a lower cost of production should be pursued. In this context, the crude extract ofJuncus maritimusLam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed the isolation and identification of the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (50% effective concentration [EC50] = 1.35 µM). This antiviral activity was confirmed with an HCV subgenomic replicon, and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. Noin vitrotoxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit RNA replication of two frequent daclatasvir-resistant mutants (L31M or Y93H in NS5A). Finally, mutants resistant to dehydrojuncusol were obtained and showed that the HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted fromJ. maritimus, inhibits infection of different HCV genotypes by targeting the NS5A protein and is active against resistant HCV variants frequently found in patients with treatment failure.IMPORTANCETens of millions of people are infected with hepatitis C virus (HCV) worldwide. Recently marketed direct-acting antivirals (DAAs) targeting HCV proteins have enhanced the efficacy of treatment. However, due to its high cost, this new therapy is not accessible to the vast majority of infected patients. Furthermore, treatment failures have also been reported due to the appearance of viral resistance. Here, we report on the identification of a new HCV inhibitor, dehydrojuncusol, that targets HCV NS5A and is able to inhibit RNA replication of replicons harboring resistance mutations to anti-NS5A DAAs used in current therapy. Dehydrojuncusol is a natural compound isolated fromJuncus maritimus, a halophilic plant species that is very common in coastlines worldwide. This molecule might serve as a lead for the development of a new therapy that is more accessible to hepatitis C patients in the future.

scholarly journals Hepatitis C virus NS5A: tales of a promiscuous protein

2004 ◽  
Vol 85 (9) ◽  
pp. 2485-2502 ◽  
Author(s):  
Andrew Macdonald ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Rna Replication ◽  
Viral Rna ◽  
Cell Physiology ◽  
Subgenomic Replicon ◽  
Cellular Environment ◽  
Cellular Context

The non-structural 5A (NS5A) protein of hepatitis C virus (HCV) has been the subject of intensive research over the last decade. It is generally accepted that NS5A is a pleiotropic protein with key roles in both viral RNA replication and modulation of the physiology of the host cell. Our understanding of the role of NS5A in the virus life cycle has been hampered by the lack of a robust in vitro system for the study of HCV replication, although the recent development of the subgenomic replicon has at least allowed us to begin to dissect the involvement of NS5A in the process of viral RNA replication. Early studies into the effects of NS5A on cell physiology relied on expression of NS5A either alone or in the context of other non-structural proteins; the advent of the replicon system has allowed the extrapolation of these studies to a more physiologically relevant cellular context. Despite recent progress, this field is controversial, and there is much work to be accomplished before we fully understand the many functions of this protein. In this article, the current state of our knowledge of NS5A, discussing in detail its direct involvement in virus replication, together with its role in modulating the cellular environment to favour virus replication and persistence, are reviewed. The effects of NS5A on interferon signalling, and the regulation of cell growth and apoptosis are highlighted, demonstrating that this protein is indeed of critical importance for HCV and is worthy of further investigation.

scholarly journals Nucleotide requirements at positions +1 to +4 for the initiation of hepatitis C virus positive-strand RNA synthesis

2011 ◽  
Vol 92 (5) ◽  
pp. 1082-1086 ◽  
Author(s):  
Udvitha Nandasoma ◽  
Christopher McCormick ◽  
Stephen Griffin ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Virus ◽  
Hammerhead Ribozyme ◽  
Rna Replication ◽  
Genome Replication ◽  
Wild Type ◽  
Subgenomic Replicon ◽  
Positive Strand ◽  
Positive Strand Rna

RNA virus genome replication requires initiation at the precise terminus of the template RNA. To investigate the nucleotide requirements for initiation of hepatitis C virus (HCV) positive-strand RNA replication, a hammerhead ribozyme was inserted at the 5′ end of an HCV subgenomic replicon, allowing the generation of replicons with all four possible nucleotides at position 1. This analysis revealed a preference for a purine nucleotide at this position for initiation of RNA replication. The sequence requirements at positions 2–4 in the context of the J6/JFH-1 virus were also examined by selecting replication-competent virus from a pool containing randomized residues at these positions. There was strong selection for both the wild-type cytosine at position 2, and the wild-type sequence at positions 2–4 (CCU). An adenine residue was well tolerated at positions 3 and 4, which suggests that efficient RNA replication is less dependent on these residues.

Hepatitis A protein VP1-2A reduced cell viability in Huh-7 cells with hepatitis C virus subgenomic RNA replication

2006 ◽  
Vol 21 (3) ◽  
pp. 625-626 ◽  
Author(s):  
Tatsuo Kanda ◽  
Osamu Yokosuka ◽  
Fumio Imazeki ◽  
Keiichi Fujiwara ◽  
Hiromitsu Saisho
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Viability ◽  
Hepatitis A ◽  
Rna Replication ◽  
Subgenomic Rna

Anti-Bovine Viral Diarrhoea Virus and Hepatitis C Virus Activity of the Cyclooxygenase Inhibitor SC-560

2009 ◽  
Vol 20 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Mika Okamoto ◽  
Masashi Sakai ◽  
Yukinori Goto ◽  
Mohammed TA Salim ◽  
Chiaki Baba ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Rna Synthesis ◽  
Bovine Viral Diarrhoea Virus ◽  
Inhibitory Effect ◽  
Host Cells ◽  
Bovine Viral Diarrhoea ◽  
Diarrhoea Virus ◽  
Mdbk Cells

Background: A number of compounds were examined for their inhibitory effect on bovine viral diarrhoea virus (BVDV) replication in cell cultures and found that some cyclooxygenase (COX) inhibitors had antiviral activity against the virus. Methods: Determination of compounds for their anti-BVDV activity was on the basis of the inhibition of virus-induced cytopathogenicity in Mardin–Darby bovine kidney (MDBK) cells. Anti-hepatitis C virus (HCV) activity was assessed by the inhibition of viral RNA synthesis in the subgenomic HCV RNA replicon cells. Results: Among the test compounds, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1 H-pyrazole (SC-560) was the most active against BVDV, and its 50% effective and cytotoxic concentrations were 10.9 ±2.8 and 93.9 ±24.5 μM in virus and mock-infected MDBK cells, respectively. The compound also suppressed BVDV RNA synthesis in a dose-dependent fashion. Studies on the mechanism of action revealed that SC-560 did not interfere with viral entry to the host cells. Furthermore, it was assumed that the antiviral activity of SC-560 was not associated with its inhibitory effect on COX. The combination of SC-560 and interferon-α was additive to synergistic in inhibiting BVDV replication. More importantly, the compound proved to be a selective inhibitor of HCV replication. Conclusions: SC-560 and its derivative might have potential as novel antiviral agents against HCV.

scholarly journals A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

2009 ◽  
Vol 83 (20) ◽  
pp. 10788-10796 ◽  
Author(s):  
Mair Hughes ◽  
Sarah Gretton ◽  
Holly Shelton ◽  
David D. Brown ◽  
Christopher J. McCormick ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Assembly ◽  
Infectious Clone ◽  
Consensus Sequence ◽  
Virus Production ◽  
Rna Replication ◽  
Subgenomic Replicon ◽  
Genotype 1B

ABSTRACT We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.

scholarly journals Alternative Approaches for Efficient Inhibition of Hepatitis C Virus RNA Replication by Small Interfering RNAs

2004 ◽  
Vol 78 (7) ◽  
pp. 3436-3446 ◽  
Author(s):  
Jan Krönke ◽  
Ralf Kittler ◽  
Frank Buchholz ◽  
Marc P. Windisch ◽  
Thomas Pietschmann ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Rna Replication ◽  
Similar Degree ◽  
Hcv Rna ◽  
Target Sequence ◽  
Small Interfering Rnas ◽  
Conserved Sequence ◽  
Nontranslated Region

ABSTRACT Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It has recently been shown that HCV RNA replication is susceptible to small interfering RNAs (siRNAs), but the antiviral activity of siRNAs depends very much on their complementarity to the target sequence. Thus, the high degree of sequence diversity between different HCV genotypes and the rapid evolution of new quasispecies is a major problem in the development of siRNA-based gene therapies. For this study, we developed two alternative strategies to overcome these obstacles. In one approach, we used endoribonuclease-prepared siRNAs (esiRNAs) to simultaneously target multiple sites of the viral genome. We show that esiRNAs directed against various regions of the HCV coding sequence as well as the 5′ nontranslated region (5′ NTR) efficiently block the replication of subgenomic and genomic HCV replicons. In an alternative approach, we generated pseudotyped retroviruses encoding short hairpin RNAs (shRNAs). A total of 12 shRNAs, most of them targeting highly conserved sequence motifs within the 5′ NTR or the early core coding region, were analyzed for their antiviral activities. After the transduction of Huh-7 cells containing a subgenomic HCV replicon, we found that all shRNAs targeting sequences in domain IV or nearby coding sequences blocked viral replication. In contrast, only one of seven shRNAs targeting sequences in domain II or III had a similar degree of antiviral activity, indicating that large sections of the NTRs are resistant to RNA interference. Moreover, we show that naive Huh-7 cells that stably expressed certain 5′ NTR-specific shRNAs were largely resistant to a challenge with HCV replicons. These results demonstrate that the retroviral transduction of HCV-specific shRNAs provides a new possibility for antiviral intervention.

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