Hepatitis C virus inhibits IL-1β-induced IκBζ expression in a Calpain- and MK2-dependent manner resulting in LCN2 suppression

Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing in vitro-transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.

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Hepatitis C Virus Core Protein Induces Lipid Droplet Redistribution in a Microtubule- and Dynein-Dependent Manner

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10.1111/j.1600-0854.2008.00767.x ◽

2008 ◽

Vol 9 (8) ◽

pp. 1268-1282 ◽

Cited By ~ 150

Author(s):

Steeve Boulant ◽

Mark W. Douglas ◽

Laura Moody ◽

Agata Budkowska ◽

Paul Targett-Adams ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Droplet ◽

Core Protein ◽

Dependent Manner ◽

Virus Core

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Human MxB Inhibits the Replication of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.01285-18 ◽

2018 ◽

Vol 93 (1) ◽

Cited By ~ 7

Author(s):

Dong-Rong Yi ◽

Ni An ◽

Zhen-Long Liu ◽

Feng-Wen Xu ◽

Kavita Raniga ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Dengue Virus ◽

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Rna Replication ◽

Encephalitis Virus ◽

Common Mechanism ◽

Type I ◽

Dependent Manner

ABSTRACTType I interferon (IFN) inhibits viruses by inducing the expression of antiviral proteins. The IFN-induced myxovirus resistance B (MxB) protein has been reported to inhibit a limited number of viruses, including HIV-1 and herpesviruses, but its antiviral coverage remains to be explored further. Here we show that MxB interferes with RNA replication of hepatitis C virus (HCV) and significantly inhibits viral replication in a cyclophilin A (CypA)-dependent manner. Our data further show that MxB interacts with the HCV protein NS5A, thereby impairing NS5A interaction with CypA and NS5A localization to the endoplasmic reticulum, two events essential for HCV RNA replication. Interestingly, we found that MxB significantly inhibits two additional CypA-dependent viruses of theFlaviviridaefamily, namely, Japanese encephalitis virus and dengue virus, suggesting a potential link between virus dependence on CypA and virus susceptibility to MxB inhibition. Collectively, these data have identified MxB as a key factor behind IFN-mediated suppression of HCV infection, and they suggest that other CypA-dependent viruses may also be subjected to MxB restriction.IMPORTANCEViruses of theFlaviviridaefamily cause major illness and death around the world and thus pose a great threat to human health. Here we show that IFN-inducible MxB restricts several members of theFlaviviridae, including HCV, Japanese encephalitis virus, and dengue virus. This finding not only suggests an active role of MxB in combating these major pathogenic human viruses but also significantly expands the antiviral spectrum of MxB. Our study further strengthens the link between virus dependence on CypA and susceptibility to MxB restriction and also suggests that MxB may employ a common mechanism to inhibit different viruses. Elucidating the antiviral functions of MxB advances our understanding of IFN-mediated host antiviral defense and may open new avenues to the development of novel antiviral therapeutics.

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NS3 protein of Hepatitis C virus associates with the tumour suppressor p53 and inhibits its function in an NS3 sequence-dependent manner

Journal of General Virology ◽

10.1099/vir.0.81735-0 ◽

2006 ◽

Vol 87 (6) ◽

pp. 1703-1713 ◽

Cited By ~ 60

Author(s):

Lin Deng ◽

Motoko Nagano-Fujii ◽

Motofumi Tanaka ◽

Yuki Nomura-Takigawa ◽

Masanori Ikeda ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Subcellular Localization ◽

Serine Protease ◽

Protease Activity ◽

Hcv Rna ◽

Dependent Manner ◽

Diffuse Type ◽

Serine Protease Activity ◽

Tumour Suppressor P53

The N-terminal 198 residues of NS3 (NS3-N) of Hepatitis C virus (HCV) subtype 1b obtained from 29 patients, as well as full-length NS3 (NS3-Full), were analysed for their subcellular localization, interaction with the tumour suppressor p53 and serine protease activity in the presence and absence of the viral cofactor NS4A. Based on the subcellular-localization patterns in the absence of NS4A, NS3-N sequences were classified into three groups, with each group exhibiting either dot-like, diffuse or a mixed type of localization. Chimeric NS3-Full sequences, each consisting of an individual NS3-N and a shared C-terminal sequence, showed the same localization patterns as those of the respective NS3-N. Site-directed mutagenesis experiments revealed that a single or a few amino acid substitutions at a particular position(s) of NS3-N altered the localization pattern. Interestingly, NS3 of the dot-like type, either NS3-N or NS3-Full, interacted with p53 more strongly than that of the diffuse type, in both the presence and the absence of NS4A. Moreover, NS3-N of the dot-like type suppressed trans-activating activity of p53 more strongly than that of the diffuse type. Serine protease activity did not differ significantly between the two types of NS3. In HCV RNA replicon-harbouring cells, physical interaction between NS3 and p53 was observed consistently and p53-mediated transcriptional activation was suppressed significantly compared with HCV RNA-negative control cells. Our results collectively suggest the possibility that NS3 plays an important role in the hepatocarcinogenesis of HCV by interacting differentially with p53 in an NS3 sequence-dependent manner.

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

Journal of Virology ◽

10.1128/jvi.73.2.1649-1654.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 1649-1654 ◽

Cited By ~ 172

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.

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Generation of Infectious Hepatitis C Virus in Immortalized Human Hepatocytes

Journal of Virology ◽

10.1128/jvi.80.9.4633-4639.2006 ◽

2006 ◽

Vol 80 (9) ◽

pp. 4633-4639 ◽

Cited By ~ 71

Author(s):

Tatsuo Kanda ◽

Arnab Basu ◽

Robert Steele ◽

Takaji Wakita ◽

Jan S. Ryerse ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Expression ◽

Culture Media ◽

Human Hepatocytes ◽

Full Length ◽

Dependent Manner ◽

Virus Growth ◽

Hcv Genotype

ABSTRACT Progress in understanding hepatitis C virus (HCV) biology has remained a challenge due to the lack of an efficient cell culture system for virus growth. In this study, we examined HCV core protein-mediated immortalized human hepatocytes (IHH) for growth of HCV. In vitro-transcribed full-length RNA from HCV genotype 1a (clone H77) was introduced into IHH by electroporation. Reverse transcription-PCR of cellular RNA isolated from HCV genome-transfected IHH suggested that viral RNA replication occurred. IHH transfected with the full-length HCV genome also displayed viral protein expression by indirect immunofluorescence. In contrast, cells transfected with polymerase-defective HCV (H77/GND) RNA as a negative control did not exhibit expression of the viral genome. Immunogold labeling demonstrated localization of E1 protein in the rough endoplasmic reticulum of RNA-transfected IHH. Virus-like particles of ∼50 nm were observed in the cytoplasm. After being inoculated with culture media of cells transfected with the full-length HCV genome, naïve IHH displayed NS5a protein expression in a dilution-dependent manner, but expression of NS5a was inhibited by prior incubation of culture medium with HCV-infected patient sera. NS5a-positive immunofluorescence of cell culture media of IHH transfected with full-length H77 RNA yielded ∼4.5 × 104 to 1 × 105 focus-forming units/ml. A similar level of virus growth was observed upon transfection of RNA from HCV genotype 2a (JFH1) into IHH. Taken together, our results suggest that IHH support HCV genome replication and virus assembly.

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Hepatitis C Virus NS5A Physically Associates with p53 and Regulates p21/waf1 Gene Expression in a p53-Dependent Manner

Journal of Virology ◽

10.1128/jvi.75.3.1401-1407.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1401-1407 ◽

Cited By ~ 177

Author(s):

Mainak Majumder ◽

Asish K. Ghosh ◽

Robert Steele ◽

Ranjit Ray ◽

Ratna B. Ray

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Transcriptional Activation ◽

Transcriptional Repression ◽

Dependent Manner ◽

Downstream Effector ◽

Transcriptional Modulation ◽

P21 Waf1 ◽

Two Hybrid

ABSTRACT We have previously demonstrated that hepatitis C virus (HCV) NS5A protein promotes cell growth and transcriptionally regulates the p21/waf1 promoter, a downstream effector gene of p53. In this study, we investigated the molecular mechanism of NS5A-mediated transcriptional repression of p21/waf1. We observed that transcriptional repression of the p21/waf1 gene by NS5A is p53 dependent by using p53 wild-type (+/+) and null (−/−) cells. Interestingly, p53-mediated transcriptional activation from a synthetic promoter containing multiple p53 binding sites (PG13-LUC) was abrogated following expression of HCV NS5A. Additional studies using pull-down experiments, in vivo coimmunoprecipitation, and mammalian two-hybrid assays demonstrated that NS5A physically associates with p53. Confocal microscopy revealed sequestration of p53 in the perinuclear membrane and colocalization with NS5A in transfected HepG2 and Saos-2 cells. Together these results suggest that an association of NS5A and p53 allows transcriptional modulation of the p21/waf1 gene and may contribute to HCV-mediated pathogenesis.

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MK-571, a Cysteinyl Leukotriene Receptor 1 Antagonist, Inhibits Hepatitis C Virus Replication

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02078-19 ◽

2020 ◽

Vol 64 (6) ◽

Cited By ~ 1

Author(s):

Isaac Ruiz ◽

Quentin Nevers ◽

Eva Hernández ◽

Nazim Ahnou ◽

Rozenn Brillet ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Hepatoma Cell ◽

Hcv Rna ◽

Dependent Manner ◽

Cysteinyl Leukotriene ◽

Leukotriene Receptor ◽

Cysteinyl Leukotriene Receptor ◽

Dose Dependent ◽

Rna Levels

ABSTRACT The quinoline MK-571 is the most commonly used inhibitor of multidrug resistance protein-1 (MRP-1) but was originally developed as a cysteinyl leukotriene receptor 1 (CysLTR1) antagonist. While studying the modulatory effect of MRP-1 on anti-hepatitis C virus (HCV) direct-acting antiviral (DAA) efficiency, we observed an unexpected anti-HCV effect of compound MK-571 alone. This anti-HCV activity was characterized in Huh7.5 cells stably harboring a subgenomic genotype 1b replicon. A dose-dependent decrease of HCV RNA levels was observed upon MK-571 administration, with a 50% effective concentration (EC50 ± standard deviation) of 9 ± 0.3 μM and a maximum HCV RNA level reduction of approximatively 1 log10. MK-571 also reduced the replication of the HCV full-length J6/JFH1 model in a dose-dependent manner. However, probenecid and apigenin homodimer (APN), two specific inhibitors of MRP-1, had no effect on HCV replication. In contrast, the CysLTR1 antagonist SR2640 increased HCV-subgenomic replicon (SGR) RNA levels in a dose-dependent manner, with a maximum increase of 10-fold. In addition, a combination of natural CysLTR1 agonist (LTD4) or antagonists (zafirlukast, cinalukast, and SR2640) with MK-571 completely reversed its antiviral effect, suggesting its anti-HCV activity is related to CysLTR1 rather to MRP-1 inhibition. In conclusion, we showed that MK-571 inhibits HCV replication in hepatoma cell cultures by acting as a CysLTR1 receptor antagonist, thus unraveling a new host-virus interaction in the HCV life cycle.

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Genetic Variants in the Promoter Region of the Macrophage Migration Inhibitory Factor are Associated with the Severity of Hepatitis C Virus-Induced Liver Fibrosis

International Journal of Molecular Sciences ◽

10.3390/ijms20153753 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3753 ◽

Cited By ~ 1

Author(s):

Wirtz ◽

Fischer ◽

Backhaus ◽

Bergmann ◽

Brandt ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Liver Fibrosis ◽

Migration Inhibitory Factor ◽

Promoter Region ◽

Macrophage Migration Inhibitory Factor ◽

Inhibitory Factor ◽

Macrophage Migration ◽

Dependent Manner ◽

Microsatellite Repeats

Two polymorphisms in the promoter region of macrophage migration inhibitory factor (MIF) - rs755622 and rs5844572 - exhibit prognostic relevance in inflammatory diseases. The aim of this study was to investigate a correlation between these MIF promoter polymorphisms and the severity of hepatitis C virus (HCV)-induced liver fibrosis. Our analysis included two independent patient cohorts with HCV-induced liver fibrosis (504 and 443 patients, respectively). The genotype of the single nucleotide polymorphism (SNP) -173 G/C and the repeat number of the microsatellite polymorphism -794 CATT5–8 were determined in DNA samples and correlated with fibrosis severity. In the first cohort, homozygous carriers of the C allele in the rs755622 had lower fibrosis stages compared to heterozygous carriers or wild types (1.25 vs. 2.0 vs. 2.0; p = 0.03). Additionally, ≥7 microsatellite repeats were associated with lower fibrosis stages (<F2) (p = 0.04). Comparable tendencies were observed in the second independent cohort, where fibrosis was assessed using transient elastography. However, once cirrhosis had been established, the C/C genotype and higher microsatellite repeats correlated with impaired liver function and a higher prevalence of hepatocellular carcinoma. Our study demonstrates that specific MIF polymorphisms are associated with disease severity and complications of HCV-induced fibrosis in a stage- and context-dependent manner.

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