scholarly journals Hepatitis C Virus (HCV) Constitutively Activates STAT-3 via Oxidative Stress: Role of STAT-3 in HCV Replication

2005 ◽  
Vol 79 (3) ◽  
pp. 1569-1580 ◽  
Author(s):  
Gulam Waris ◽  
James Turkson ◽  
Tarek Hassanein ◽  
Aleem Siddiqui
Keyword(s):  
Oxidative Stress ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Hcv Rna ◽  
Pyrrolidine Dithiocarbamate ◽  
Constitutive Activation ◽  
Mitogen Activated Protein

ABSTRACT The hepatitis C virus (HCV) causes chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. We have previously shown that HCV nonstructural proteins induce activation of STAT-3 via oxidative stress and Ca2+ signaling (G. Gong, G. Waris, R. Tanveer, and A. Siddiqui, Proc. Natl. Acad. Sci. USA 98:9599-9604, 2001). In this study, we focus on the signaling pathway leading to STAT-3 activation in response to oxidative stress induced by HCV translation and replication activities. Here, we demonstrate the constitutive activation of STAT-3 in HCV replicon-expressing cells. The HCV-induced STAT-3 activation was inhibited in the presence of antioxidant (pyrrolidine dithiocarbamate) and Ca2+ chelators (BAPTA-AM and TMB-8). Previous studies have shown that maximum STAT-3 transactivation requires Ser727 phosphorylation in addition to tyrosine phosphorylation. Using a series of inhibitors and dominant negative mutants, we show that HCV-induced activation of STAT-3 is mediated by oxidative stress and influenced by the activation of cellular kinases, including p38 mitogen-activated protein kinase, JNK, JAK-2, and Src. Our results also suggest a potential role of STAT-3 in HCV RNA replication. We also observed the constitutive activation of STAT-3 in the liver biopsy of an HCV-infected patient. These studies provide an insight into the mechanisms by which HCV induces intracellular events relevant to liver pathogenesis associated with the viral infection.

scholarly journals Genotype Dependence of Hepatitis C Virus Load Measurement in Commercially Available Quantitative Assays

1999 ◽  
Vol 37 (8) ◽  
pp. 2525-2532 ◽  
Author(s):  
Janet Mellor ◽  
Anna Hawkins ◽  
Peter Simmonds
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Narrow Range ◽  
Antiviral Treatment ◽  
Hcv Rna ◽  
Clinical Specimens ◽  
Rna Transcripts ◽  
Relative Quantitation ◽  
Version 2.0

Standardization and genotype independence of methods used to quantify hepatitis C virus (HCV) RNA in clinical specimens are necessary for accurate assessment of the role of HCV quantitation as a prognostic marker for HCV infection and monitoring of the response to antiviral treatment. Commercially available methods used to measure HCV loads include PCR-based (Roche Monitor) and hybridization-based (Quantiplex bDNA-2) methods. Recently, a new version of the Roche Monitor assay (version 2.0) has become available; it has been modified to achieve more equal quantitation of different HCV genotypes. Consistent with previous reports, Roche Monitor version 1.0 substantially underestimated concentrations of RNA transcripts of types 2b, 3a, 4a, 5a, and 6a and virus loads in individuals infected with genotypes 2 to 6 relative to reference tests. However, version 2.0 achieved equivalent quantitation of each genotype over a narrow quantitative range (103 to 5 × 105 copies of RNA/ml) but significantly underestimated RNA concentrations above this range. The assay showed an equivalent inability to quantify high levels of HCV RNA in plasma samples, and this was responsible for the falsely narrow range of virus loads detected in HCV-infected individuals. In contrast, the Chiron bDNA-2 assay could only measure RNA concentrations in the upper quantitative range (2 × 105 to 5 × 107 copies of RNA/ml) but showed equivalent sensitivity for genotypes 1 to 5; however, concentrations of type 6a RNA transcripts and virus loads in clinical specimens from individuals infected with type 6a were underestimated by a factor of 2 to 4. Differences were observed between PCR- and hybridization-based assays in their relative quantitation of HCV RNA transcripts and HCV genomic RNA, which may cause problems with the use of transcripts for interassay calibration.

scholarly journals Arsenic Trioxide Inhibits Hepatitis C Virus RNA Replication through Modulation of the Glutathione Redox System and Oxidative Stress

2008 ◽  
Vol 83 (5) ◽  
pp. 2338-2348 ◽  
Author(s):  
Misao Kuroki ◽  
Yasuo Ariumi ◽  
Masanori Ikeda ◽  
Hiromichi Dansako ◽  
Takaji Wakita ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Arsenic Trioxide ◽  
Redox System ◽  
Rna Replication ◽  
Hcv Rna ◽  
Glutathione Redox System ◽  
And Oxidative Stress ◽  
Glutathione Redox

ABSTRACT Arsenic trioxide (ATO), a therapeutic reagent used for the treatment of acute promyelocytic leukemia, has recently been reported to increase human immunodeficiency virus type 1 infectivity. However, in this study, we have demonstrated that replication of genome-length hepatitis C virus (HCV) RNA (O strain of genotype 1b) was notably inhibited by ATO at submicromolar concentrations without cell toxicity. RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were also inhibited by ATO after the HCV infection. To clarify the mechanism of the anti-HCV activity of ATO, we examined whether or not PML is associated with this anti-HCV activity, since PML is known to be a target of ATO. Interestingly, we observed the cytoplasmic translocation of PML after treatment with ATO. However, ATO still inhibited the HCV RNA replication even in the PML knockdown cells, suggesting that PML is dispensable for the anti-HCV activity of ATO. In contrast, we found that N-acetyl-cysteine, an antioxidant and glutathione precursor, completely and partially eliminated the anti-HCV activity of ATO after 24 h and 72 h of treatment, respectively. In this context, it is worth noting that we found an elevation of intracellular superoxide anion radical, but not hydrogen peroxide, and the depletion of intracellular glutathione in the ATO-treated cells. Taken together, these findings suggest that ATO inhibits the HCV RNA replication through modulation of the glutathione redox system and oxidative stress.

scholarly journals Interactions between Viral Nonstructural Proteins and Host Protein hVAP-33 Mediate the Formation of Hepatitis C Virus RNA Replication Complex on Lipid Raft

2004 ◽  
Vol 78 (7) ◽  
pp. 3480-3488 ◽  
Author(s):  
Lu Gao ◽  
Hideki Aizaki ◽  
Jian-Wen He ◽  
Michael M. C. Lai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Raft ◽  
Critical Role ◽  
Rna Replication ◽  
Dominant Negative ◽  
Host Protein ◽  
Hcv Rna ◽  
Replication Complex ◽  
Detergent Resistant Membranes

ABSTRACT The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.

scholarly journals Participation of Rab5, an Early Endosome Protein, in Hepatitis C Virus RNA Replication Machinery

2007 ◽  
Vol 81 (9) ◽  
pp. 4551-4563 ◽  
Author(s):  
Michelle Stone ◽  
Shuaizheng Jia ◽  
Won Do Heo ◽  
Tobias Meyer ◽  
Kouacou V. Konan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fluorescent Protein ◽  
Early Endosome ◽  
Dominant Negative ◽  
Hcv Rna ◽  
Small Interfering Rnas ◽  
Subgenomic Replicon ◽  
Gfp Fluorescence ◽  
Positive Strand Rna

ABSTRACT Like most positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its genome on the surface of rearranged membranes. We have shown previously that HCV NS4AB, but not the product NS4B, inhibits endoplasmic reticulum (ER)-to-Golgi protein traffic (K. V. Konan, T. H. Giddings, Jr., M. Ikeda, K. Li, S. M. Lemon, and K. Kirkegaard, J. Virol. 77:7843-7855). However, both NS4AB and NS4B can induce “membranous web” formation, first reported by Egger et al. (D. B Egger, R. Gosert, L. Bianchi, H. E. Blum, D. Moradpour, and K. Bienz, J. Virol. 76:5974-5984), which is also observed in HCV-infected cells (Y. Rouille, F. Helle, D. Delgrange, P. Roingeard, C. Voisset, E. Blanchard, S. Belouzard, J. McKeating, A. H. Patel, G. Maertens, T. Wakita, C. Wychowski, and J. Dubuisson, J. Virol. 80:2832-2841) and cells that bear a subgenomic NS5A-green fluorescent protein (GFP) replicon (D. Moradpour, M. J. Evans, R. Gosert, Z. Yuan, H. E. Blum, S. P. Goff, B. D. Lindenbach, and C. M. Rice, J. Virol. 78:7400-7409). To determine the intracellular origin of the web, we examined NS4B colocalization with endogenous cellular markers in the context of the full-length or subgenomic replicon. We found that, in addition to ER markers, early endosome (EE) proteins, including Rab5, were associated with web-inducing protein NS4B. Furthermore, an immunoisolated fraction containing NS4B was found to contain both ER and EE proteins. Using fluorescence microscopy, we showed that wild-type and constitutively active Rab5 proteins were associated with NS4B. Interestingly, expression of dominant-negative Rab5 resulted in significant loss of GFP fluorescence in NS5A-GFP replicon cells. We also found that a small reduction in Rab5 protein expression decreased HCV RNA synthesis significantly. Furthermore, transfection of labeled Rab5 small interfering RNAs into NS5A-GFP replicon cells resulted in a significant decrease in GFP fluorescence. Finally, Rab5 protein was found to coimmunoprecipitate with HCV NS4B. These studies suggest that EE proteins, including Rab5, may play a role in HCV genome replication or web formation.

scholarly journals The Role of the RNA-RNA Interactome in the Hepatitis C Virus Life Cycle

2020 ◽  
Vol 21 (4) ◽  
pp. 1479 ◽  
Author(s):  
Cristina Romero-López ◽  
Alfredo Berzal-Herranz
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Long Range ◽  
Genomic Sequence ◽  
Rna Virus ◽  
Hcv Rna ◽  
Structural Elements ◽  
Control Translation ◽  
Rna Interaction

RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.

scholarly journals The Mitogen-Activated Protein Kinase BcSak1 of Botrytis cinerea Is Required for Pathogenic Development and Has Broad Regulatory Functions Beyond Stress Response

2012 ◽  
Vol 25 (6) ◽  
pp. 802-816 ◽  
Author(s):  
Jens Heller ◽  
Nadja Ruhnke ◽  
José Juan Espino ◽  
Michelli Massaroli ◽  
Isidro Gonzalez Collado ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Protein Kinase ◽  
Osmotic Stress ◽  
Botrytis Cinerea ◽  
Fluorescent Protein ◽  
Mitogen Activated Protein Kinase ◽  
In Planta ◽  
Mitogen Activated Protein

The mitogen-activated protein kinase (MAPK) BcSak1 of Botrytis cinerea is activated upon exposure to H2O2 and, hence, might be involved in coping with oxidative stress during infection. However, beside osmotic and oxidative stress sensitivity, Δbcsak1 mutants have a pleiotropic phenotype, as they do not produce conidia and are unable to penetrate unwounded host tissue. In this study, the role of BcSak1 was investigated in the stress response and during infection of French beans by Botrytis cinerea. Using a macroarray approach, it was shown that BcSak1 is only marginally involved in the specific oxidative stress response. In fact, the induction of several genes after oxidative stress treatment is BcSak1-dependent, but most of these genes are also induced under conditions of osmotic stress. The majority of genes regulated by BcSak1 are not involved in the stress response at all. Using a translational fusion of BcSak1 to green fluorescent protein, it was shown clearly that the localization of this MAPK depends on the type of stress being applied; it associates rapidly to the nucleus only under osmotic stress. Therefore, a model is proposed in which BcSak1 acts in the cytosol by activation of one or more transcription factors under oxidative stress and, at the same time, it reacts to osmotic stress by migrating to the nucleus. Interestingly, the MAPK is also involved in the regulation of secondary metabolism, as the major phytotoxins secreted by this fungus are reduced in the Δbcsak1 deletion mutant. Experiments done in planta underlined the essential role of BcSak1 in the early stages of infection, when it translocates to the nucleus and then changes to cytosolic distribution during hyphal growth within the tissue.

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