scholarly journals Hepatitis C Virus NS5A-Mediated Activation of Phosphoinositide 3-Kinase Results in Stabilization of Cellular β-Catenin and Stimulation of β-Catenin-Responsive Transcription

2005 ◽  
Vol 79 (8) ◽  
pp. 5006-5016 ◽  
Author(s):  
Andrew Street ◽  
Andrew Macdonald ◽  
Christopher McCormick ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Glycogen Synthase ◽  
Recombinant Baculovirus ◽  
Contributory Factor ◽  
Forkhead Transcription Factor ◽  
Human Hepatoma Cells ◽  
Gsk 3Β ◽  
Phosphoinositide 3 Kinase ◽  
Stimulation Of

ABSTRACT The hepatitis C virus (HCV) nonstructural NS5A protein has been shown to bind to and activate phosphoinositide 3-kinase (PI3K), resulting in activation of the downstream effector serine/threonine kinase Akt/protein kinase B. Here we present data pertaining to the effects of NS5A-mediated Akt activation on its downstream targets. Using a recombinant baculovirus to deliver the complete HCV polyprotein to human hepatoma cells in a tetracycline-regulable fashion, we confirm that expression of the complete HCV polyprotein also activates PI3K and Akt. We further show that this results in the inhibition of the Akt substrate Forkhead transcription factor and the stimulation of phosphorylation of a second key Akt substrate, glycogen synthase kinase-3β (GSK-3β). Phosphorylation of GSK-3β results in its inactivation; consistent with this, we show that expression of the HCV polyprotein results in the accumulation of β-catenin. Finally, we show that levels of β-catenin-dependent transcription are also elevated in the presence of the HCV polyprotein. Given the prevalence of β-catenin mutations in many human tumors, especially colon and hepatocellular carcinomas, these data implicate NS5A-mediated PI3K activation as a contributory factor in the increasingly common association between HCV infection and the development of hepatocellular carcinoma.

Parallel microRNA and mRNA expression profiling of (genotype 1b) human hepatoma cells expressing hepatitis C virus

2010 ◽  
Vol 30 (10) ◽  
pp. 1490-1504 ◽  
Author(s):  
Nury M. Steuerwald ◽  
Judith C. Parsons ◽  
Kristen Bennett ◽  
Tonya C. Bates ◽  
Herbert L. Bonkovsky
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mrna Expression ◽  
Expression Profiling ◽  
Hepatoma Cells ◽  
Human Hepatoma ◽  
Human Hepatoma Cells ◽  
Genotype 1B ◽  
Mrna Expression Profiling

scholarly journals Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3

2012 ◽  
Vol 93 (2) ◽  
pp. 247-258 ◽  
Author(s):  
Anna Pasetto ◽  
Lars Frelin ◽  
Anette Brass ◽  
Anila Yasmeen ◽  
Sarene Koh ◽  
...  
Keyword(s):  
T Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Cytotoxic T Lymphocyte ◽  
Hcv Rna ◽  
High Avidity ◽  
Immune Recognition ◽  
Structural Protein ◽  
Human Hepatoma Cells

Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell–BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS31073–1081 CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS31073 peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

scholarly journals Analysis of Antigenicity and Topology of E2 Glycoprotein Present on Recombinant Hepatitis C Virus-Like Particles

2002 ◽  
Vol 76 (15) ◽  
pp. 7672-7682 ◽  
Author(s):  
Reginald F. Clayton ◽  
Ania Owsianka ◽  
Jim Aitken ◽  
Susan Graham ◽  
David Bhella ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Function Analysis ◽  
Recombinant Baculovirus ◽  
Microscopic Analysis ◽  
Surface Topology ◽  
Viral Glycoprotein ◽  
Electron Microscopic ◽  
Virus Like Particles

ABSTRACT Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2660) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2660 with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2660 but not VLPs or FL E1E2 complex. The antigenic differences between E2660, FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.

scholarly journals TNRC6 proteins modulate hepatitis C virus replication by spatially regulating the binding of miR-122/Ago2 complexes to viral RNA

2019 ◽  
Vol 47 (12) ◽  
pp. 6411-6424 ◽  
Author(s):  
You Li ◽  
Li Wang ◽  
Efraín E Rivera-Serrano ◽  
Xian Chen ◽  
Stanley M Lemon
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Spatial Organization ◽  
Host Factor ◽  
Viral Rna ◽  
Fine Tuning ◽  
Hcv Rna ◽  
Human Hepatoma Cells ◽  
Translational Activity ◽  
The Stability

AbstractThe liver-specific microRNA, miR-122, is an essential host factor for replication of the hepatitis C virus (HCV). miR-122 stabilizes the positive-strand HCV RNA genome and promotes its synthesis by binding two sites (S1 and S2) near its 5′ end in association with Ago2. Ago2 is essential for both host factor activities, but whether other host proteins are involved is unknown. Using an unbiased quantitative proteomics screen, we identified the TNRC6 protein paralogs, TNRC6B and TNRC6C, as functionally important but redundant components of the miR-122/Ago2 host factor complex. Doubly depleting TNRC6B and TNRC6C proteins reduced HCV replication in human hepatoma cells, dampening miR-122 stimulation of viral RNA synthesis without reducing the stability or translational activity of the viral RNA. TNRC6B/C were required for optimal miR-122 host factor activity only when S1 was able to bind miR-122, and restricted replication when S1 was mutated and only S2 bound by miR-122. TNRC6B/C preferentially associated with S1, and TNRC6B/C depletion enhanced Ago2 association at S2. Collectively, these data suggest a model in which TNRC6B/C regulate the assembly of miR-122/Ago complexes on HCV RNA, preferentially directing miR-122/Ago2 to S1 while restricting its association with S2, thereby fine-tuning the spatial organization of miR-122/Ago2 complexes on the viral genome.

scholarly journals Th1 Disabled Function in Response to TLR4 Stimulation of Monocyte-Derived DC from Patients Chronically-Infected by Hepatitis C Virus

PLoS ONE ◽  
2008 ◽  
Vol 3 (5) ◽  
pp. e2260 ◽  
Author(s):  
Laure Perrin-Cocon ◽  
Sophie Agaugué ◽  
Olivier Diaz ◽  
Béatrice Vanbervliet ◽  
Sandra Dollet ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Stimulation Of

scholarly journals Insulin directly stimulates mitochondrial glucose oxidation in the heart

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Qutuba G. Karwi ◽  
Cory S. Wagg ◽  
Tariq R. Altamimi ◽  
Golam M. Uddin ◽  
Kim L. Ho ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Glucose Oxidation ◽  
Glycogen Synthase ◽  
Acid Oxidation ◽  
Mouse Heart ◽  
Insulin Stimulation ◽  
Direct Stimulation ◽  
Gsk 3Β ◽  
Stimulation Of

Abstract Background Glucose oxidation is a major contributor to myocardial energy production and its contribution is orchestrated by insulin. While insulin can increase glucose oxidation indirectly by enhancing glucose uptake and glycolysis, it also directly stimulates mitochondrial glucose oxidation, independent of increasing glucose uptake or glycolysis, through activating mitochondrial pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. However, how insulin directly stimulates PDH is not known. To determine this, we characterized the impacts of modifying mitochondrial insulin signaling kinases, namely protein kinase B (Akt), protein kinase C-delta (PKC-δ) and glycogen synthase kinase-3 beta (GSK-3β), on the direct insulin stimulation of glucose oxidation. Methods We employed an isolated working mouse heart model to measure the effect of insulin on cardiac glycolysis, glucose oxidation and fatty acid oxidation and how that could be affected when mitochondrial Akt, PKC-δ or GSK-3β is disturbed using pharmacological modulators. We also used differential centrifugation to isolate mitochondrial and cytosol fraction to examine the activity of Akt, PKC-δ and GSK-3β between these fractions. Data were analyzed using unpaired t-test and two-way ANOVA. Results Here we show that insulin-stimulated phosphorylation of mitochondrial Akt is a prerequisite for transducing insulin’s direct stimulation of glucose oxidation. Inhibition of mitochondrial Akt completely abolishes insulin-stimulated glucose oxidation, independent of glucose uptake or glycolysis. We also show a novel role of mitochondrial PKC-δ in modulating mitochondrial glucose oxidation. Inhibition of mitochondrial PKC-δ mimics insulin stimulation of glucose oxidation and mitochondrial Akt. We also demonstrate that inhibition of mitochondrial GSK3β phosphorylation does not influence insulin-stimulated glucose oxidation. Conclusion We identify, for the first time, insulin-stimulated mitochondrial Akt as a prerequisite transmitter of the insulin signal that directly stimulates cardiac glucose oxidation. These novel findings suggest that targeting mitochondrial Akt is a potential therapeutic approach to enhance cardiac insulin sensitivity in condition such as heart failure, diabetes and obesity.

scholarly journals Hepatitis C Virus Core Protein Down-Regulates p21Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cells

PLoS ONE ◽  
2013 ◽  
Vol 8 (4) ◽  
pp. e61089 ◽  
Author(s):  
Tzu-Yue Shiu ◽  
Shih-Ming Huang ◽  
Yu-Lueng Shih ◽  
Heng-Cheng Chu ◽  
Wei-Kuo Chang ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Hepatoma Cells ◽  
Human Hepatoma ◽  
Human Hepatoma Cells ◽  
Virus Core ◽  
Induced Apoptosis

scholarly journals Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Elena Moreno ◽  
Isabel Gallego ◽  
Josep Gregori ◽  
Adriana Lucía-Sanz ◽  
María Eugenia Soria ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Evolution ◽  
Progeny Production ◽  
Human Hepatoma Cells ◽  
Cellular Environment ◽  
Mutant Spectrum ◽  
Phenotypic Diversification ◽  
Population Equilibrium

ABSTRACT Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment. IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments.

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