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1.
Fig 8

Fig 8. From: Low Oxygen Tension Enhances Hepatitis C Virus Replication.

Analysis of human liver biopsy specimens reveals a correlation between elevated HCV RNA amounts and increased expression of genes involved in anaerobic metabolism. (A) qRT-PCR quantification of HK2, LDH-A, CKB, and VEGF mRNA amounts in total RNA isolated from 12 liver samples (LB 1 to 12) from patients with chronic hepatitis C. Bars represent mean values from three independent repetitions, and error bars indicate standard deviations. YWHAZ mRNA levels were used for normalization. (B) HCV positive-strand RNA copy levels from the same samples, as quantified using the branched DNA assay. Values were normalized to those obtained with sample LB 1.

N. Vassilaki, et al. J Virol. 2013 March;87(5):2935-2948.
2.
Fig 1

Fig 1. From: Low Oxygen Tension Enhances Hepatitis C Virus Replication.

Schematic representation of HCV constructs used in this study. From top to bottom: JFH1 wild-type (wt) virus genome; JFH1/adpt1, cell culture-adapted JFH1 genome containing virus titer-enhancing mutations (indicated with asterisks) (12); Jc1, chimeric virus genome composed of the J6CF structural and JFH1 nonstructural regions (gray and white boxes, respectively); JcR2a, a Jc1 derivative containing the R-Luc gene (striped box) fused N-terminally to 16 codons of the core gene (white box) and C-terminally to the FMDV 2A protease-coding sequence (gray striped box); LucCore-NSJFH1, a bicistronic replicon encoding a firefly luciferase (F-Luc)-ubiquitin (Ub)-core fusion protein (N-terminal 161 residues of the H77 isolate); Luc-NSJFH1/delGDD, containing a three-codon deletion in the NS5B region (delGDD) gene. Black bars in all panels indicate HCV NTRs. EI, IRES of the encephalomyocarditis virus.

N. Vassilaki, et al. J Virol. 2013 March;87(5):2935-2948.
3.
Fig 7

Fig 7. From: Low Oxygen Tension Enhances Hepatitis C Virus Replication.

Three percent oxygen enhances HCV replication in Huh7.5dif cells. (A) Effect of low oxygen tension on JcR2a-infected Huh7.5dif cells that were incubated as specified at the top and harvested at various time points. Mean values are expressed relative to that obtained with cells cultured at 20→20% O2. (B) Effect of low oxygen on Jc1 RNA replication as determined by quantification of intracellular positive-strand RNA copies. Values are expressed relative to those obtained at 20→20% O2 24 h p.i. Numbers above the bars refer to ratios of RNA copies. 20→3*, cells that were transferred immediately after virus addition from 20% to 3% O2. (C) Effect of 3% O2 on GLUT1 and HK2 genes in Jc1-infected Huh7.5dif cells. mRNA amounts were quantified by qRT-PCR and expressed relative to values obtained with cells cultured at 20→20% O2 and harvested 24 h p.i. M, mock-infected cells. In all panels, bars represent mean values from at least three independent experiments in triplicate. Error bars indicate standard deviations.

N. Vassilaki, et al. J Virol. 2013 March;87(5):2935-2948.
4.
Fig 5

Fig 5. From: Low Oxygen Tension Enhances Hepatitis C Virus Replication.

Early induced reprogramming of cellular energetics by hypoxia. (A) Upregulation of VEGFA and GLUT1, HK2, ENO1, and LDH-A genes in JFH1/adpt1-infected Huh7.5 cells that were cultured at 20→3 or 3→3% O2 relative to 20→20% O2. Values were normalized to those obtained with cells kept at 20% O2 and harvested 3 h p.i. For 3→3 infected cells, values were measured only at 3, 24, and 48 h p.i. J, JFH1/adpt1-infected cells; M, mock-infected cells. (B) Three percent oxygen increases the ATP content of hepatoma cells. Intracellular ATP levels in JcR2a-infected Huh7.5 cells (1 TCID50/cell) (right), JFH1 RNA-electroporated Huh7-Lunet cells (10 μg RNA/4 × 106 cells) (middle), or mock-transfected Huh7-Lunet cells (left). Cells were incubated as specified at the top and harvested at given time points. Mean values were normalized to the ones detected in cells cultured at 20→20% O2 and harvested 24 h postinfection/posttransfection. In all panels, bars represent mean values from at least three independent experiments in triplicate or quintuplicate (ATP content). Error bars indicate standard deviations.

N. Vassilaki, et al. J Virol. 2013 March;87(5):2935-2948.
5.
Fig 2

Fig 2. From: Low Oxygen Tension Enhances Hepatitis C Virus Replication.

Low oxygen tension enhances the production of JFH1-derived viruses in Huh7.5-infected cells. (A) Schematic representation of the experimental procedure. Supernatants of Huh7-Lunet cells electroporated with JFH1/adpt1 or JcR2a RNA (10 μg/4 × 106 cells) and incubated at 20% O2 were collected and used for infection of naive Huh7.5 cells (1 TCID50/cell) that were seeded at 30% confluence (to avoid pericellular hypoxia) and preincubated for 18 h at 20% or 3% O2, respectively. Cells preincubated at 20% O2 were cultured at either 20% or 3% O2 until harvest (20→20 or 20→3, respectively). Cells preincubated at 3% O2 were incubated at 3% O2 (3→3) until harvest. (B) Indirect immunofluorescence for NS5A in JFH1/adpt1-infected cells incubated as specified on the right of the panels. Nuclei were stained with propidium iodide (PI; left column). Bar, 80 μm. A representative experiment of two independent repetitions is shown. (C) (Top) Western blot analysis of NS5A (top) in JFH1/adpt1-infected cells incubated as specified in the top of each lane. M, mock-infected cells. (Bottom) β-Actin served as a loading control. Numbers on the right refer to the positions of molecular mass marker proteins. A representative experiment of 3 independent repetitions is shown. (Bottom) Imaging quantification of NS5A signals, normalized by using β-actin signals. Quantity I Bio-Rad software was used to quantify the respective signals from the 3 independent repetitions of the Western blot analysis. Mean values are expressed relative to that obtained at 20→20% O2. (D) Enhanced release of infectious HCV at 3% O2 from JFH1/adpt1-infected Huh7.5 cells, as determined by a TCID50 assay. The gray horizontal line indicates the cutoff of the assay (∼2 TCID50/ml). A representative experiment of three independent repetitions is shown.

N. Vassilaki, et al. J Virol. 2013 March;87(5):2935-2948.
6.
Fig 9

Fig 9. From: Low Oxygen Tension Enhances Hepatitis C Virus Replication.

Low-oxygen-mediated oncogene upregulation is linked to enhancement of HCV replication. (A) Upregulation of FOS, MYC, and SRC genes in JFH1/adpt1 (J)-infected Huh7.5 or Huh7.5dif cells. M, mock-infected cells. (B) Elevated levels of AKT phosphorylation in infected Huh7.5 cells cultured under hypoxia. Western blot analysis of endogenous levels of phosphorylated (top) and total (bottom) AKT. M, mock-infected cells. A representative experiment of two independent repetitions is shown. (C) JUN-FOS overexpression enhances intracellular ATP levels and HCV replication. Huh7.5 cells cotransfected with the MMP1-Luc (0.05 μg/4 × 104 cells)- and the JUN-FOS (0.4 μg/4 × 104 cells)-encoding plasmids or the respective vector (control) were inoculated 18 h p.t. with JcR2a (1 TCID50/cell) and further incubated at 20% O2 for 24 h. JUN-FOS overexpression enhances TRE-dependent expression (left), increases HCV RNA replication (middle), and results in a detectable upregulation of the intracellular ATP (right). For all panels, values were normalized to those obtained with control-transfected cells. (D) AKT inhibition reduces hypoxia-mediated HCV enhancement. Huh7.5 cells were inoculated with JcR2a and 4 h later were untreated or treated with the AKT inhibitor VIII (Akti-1/2) at the following concentrations: C1, 2.5 μC; C2, 5 μC; C3, 10 μC. After 24 h of incubation of cells at 20% or 3% O2, luciferase activities were determined. Values were normalized to those obtained with untreated cells cultured at 20% O2. In all panels, bars represent mean values from at least three independent experiments in triplicate or quintuplicate (ATP content). Error bars indicate standard deviations.

N. Vassilaki, et al. J Virol. 2013 March;87(5):2935-2948.
7.
Fig 4

Fig 4. From: Low Oxygen Tension Enhances Hepatitis C Virus Replication.

Hypoxia-mediated enhancement of HCV replication is not facilitated by HIF-1α/2α. (A and B) HIF-1α is stabilized and HIFs are activated at 3% O2. (A) (Top) Western blot analysis of endogenous HIF-1α protein using lysates of JFH1/adpt1-infected Huh7.5 cells incubated at 20% or 3% O2. M, mock-infected cells. (Bottom) β-Actin served as a loading control. (B) HRE-dependent expression of F-Luc in Huh7.5 cells transfected with the 9xHRE-Luc construct, infected with JFH1/adpt1 (1 TCID50/cell) 18 h later, and subsequently cultured at 20% or 3% O2 until harvest. Values were normalized to those obtained with cells cultured at 20% O2. (C and D) HIF-1α and HIF-2α silencing does not affect low-oxygen-mediated enhancement of HCV replication. Huh7.5 cells cotransfected with the 9xHRE-Luc construct (0.05 μg/4 × 104 cells) and a mixture of siRNAs targeting HIF-1α and HIF-2α (20 nM each; siHIF1α/2α) or a control siRNA (40 nM) (sictrl) were inoculated with JcR2a (1 TCID50/cell) 18 h p.t. and further incubated at 20% or 3% O2. (C) (Top) Western blot analysis of endogenous HIF-1α protein. (Bottom) β-Actin served as a loading control. (D) Effect of HIF-1α/2α knockdown on the following. (Left) HIF activation, as determined by HRE-dependent F-Luc activity. (Right) HCV replication, as determined by R-Luc activity. Mean values were normalized to the reporter activity measured in cells that were cultured at 20% O2. (E and F) HIF-1α and HIF-2α overexpression is not sufficient to enhance HCV replication. Huh7.5 cells cotransfected with the 9xHRE-Luc construct (0.05 μg/4 × 104 cells) and one plasmid expressing GFP-HIF-1α, GFP-HIF-2α, or GFP (0.4 μg/4 × 104 cells) were inoculated with JcR2a (1 TCID50/cell) 18 h p.t. and subsequently incubated at 20% or 3% O2. ctrl, cells transfected with the plasmid expressing GFP. Transfection efficiency was 60 to 70%. (E) HIF-1α and -2α overexpression as determined by GFP-specific Western blot analysis. (F) Effect of HIF-1α or HIF-2α overexpression on the following. (Left) HIF activation, as determined by HRE-dependent luciferase expression. (Right) HCV replication, as determined by R-Luc expression. Mean values were normalized to the reporter activity detected in cells cultured at 20% O2. For Western blot analysis, at each time, a representative experiment of at least two independent repetitions is shown. In all panels, bars represent mean values from at least three independent experiments in triplicate. Error bars indicate standard deviations.

N. Vassilaki, et al. J Virol. 2013 March;87(5):2935-2948.
8.
Fig 6

Fig 6. From: Low Oxygen Tension Enhances Hepatitis C Virus Replication.

Replacing the glycolytic substrate with oxidative ones, as well as CKB silencing, reduces low-oxygen-mediated HCV enhancement. (A to C) Huh7.5 cells preincubated at 20% or 3% O2 were inoculated with JcR2a (1 TCID50/cell) and cultured at the given conditions in complete DMEM containing high glucose (glu h; 25 mM), low glucose (glu l; 5.56 mM), galactose instead of glucose (gal; 10 mM), or no glucose (-glu). (A) Bioluminescent measurement of intracellular ATP levels. Mean values (%) were normalized to ATP levels detected in cells cultured at 20→20% O2. (B) JcR2a-derived luciferase activity expressed as fold induction relative to values obtained with cells cultured at 20→20% O2. (C) qRT-PCR-based quantification of HK2 mRNA in cells treated as described above. Results are expressed as fold induction of gene expression relative to that in cells cultured at 20→20% O2 in DMEM with high glucose. (D) Upregulation of CKB expression at 3% O2. qRT-PCR analysis of CKB mRNA amounts in JFH1/adpt1-infected Huh7.5 cells cultured as specified on the right. Data were normalized to values obtained with cells cultured at 20→20% O2 and harvested 3 h p.i. For 3→3 infected cells, values were measured only at 3, 24, and 48 h p.i. J, JFH1/adpt1-infected cells. M, mock-infected cells. (E and F) Huh7.5 cells transfected with an siRNA targeting CKB (siCKB) or a control siRNA (sictrl) (100 nM) were inoculated 18 h p.t. with JcR2a (1 TCID50/cell), supplied with DMEM containing either high glucose or galactose (gal), and incubated at 20% O2 or transferred to 3% O2. (E) Intracellular ATP levels as determined by a bioluminescent assay. (F) JcR2a-derived luciferase activity. Values were normalized to those obtained with cells cultured at 20→20% O2 and transfected with the same siRNA. In all panels, bars represent mean values from at least three independent experiments in triplicate or quintuplicate (ATP content). Error bars indicate standard deviations.

N. Vassilaki, et al. J Virol. 2013 March;87(5):2935-2948.
9.
Fig 3

Fig 3. From: Low Oxygen Tension Enhances Hepatitis C Virus Replication.

Three percent oxygen positively regulates the HCV genome replication. (A and B) Enhanced HCV RNA replication in Huh7.5 cells infected (1 TCID50/cell) with one of two viruses. (A) JcR2a virus. Cells were cultured as specified in the upper left, and replication-derived luciferase activity is expressed as relative light units (RLU) per μg of total protein amount. Values obtained with 20→20 cells were set (each time) to one. (B) JFH1/adpt1 virus. qRT-PCR analysis of the intracellular HCV-negative (−) and -positive (+) strand RNA copies from cells incubated at 20→20, 20→3, or 3→3% O2, expressed relative to the positive-strand RNA obtained at 20→20% O2 at 24 h p.i. YWHAZ mRNA levels were used for normalization. Above the respective bars, ratios of RNA copies at 20→3 and 3→3 versus 20→20% O2 for each time point are shown. (C to F) Low oxygen tension does not affect virus entry. (C) qRT-PCR analysis of the intracellular HCV-negative (−) and -positive (+) strand RNA copies from Huh7.5 cells inoculated with JFH1/adpt1 virus (1 TCID50/cell) and incubated for 4 h as specified. 20→3* refers to cells that were preincubated at 20% O2 and transferred immediately after virus addition from 20% to 3% O2. Values (normalized as described above) are expressed relative to the positive-strand RNA obtained at 20→20% O2 at 4 h postinoculation. (D) Luciferase activities, derived from JcR2a and LucCore-NSJFH1 RNA-transfected Huh7-Lunet cells (10 and 5 μg RNA/4 × 106 cells, respectively). After transfection, the cells were further cultured at 20% or 3% O2, and luciferase activities at 3% O2 are expressed as fold inductions of those obtained at 20% O2. (E and F) Enhanced viral protein expression and release of infectivity at 3% O2 upon JFH1 RNA electroporation (10 μg RNA/4 × 106 cells) of Huh7-Lunet cells. (E) (Top) Western blot analysis of NS5A production in cells incubated at 20% or 3% O2. (Bottom) β-Actin served as a loading control. M, mock nontransfected cells. A representative experiment of two independent repetitions is shown. (F) Kinetics of release of infectivity from the viral RNA-transfected cells incubated at 20% or 3% (TCID50 assay). A representative experiment of three independent repetitions is shown. (G) Three percent oxygen does not positively affect HCV RNA expression. Huh7-Lunet cells preincubated at 20% or 3% O2 for 18 h were transfected with Luc-NSJFH1/delGDD RNA and further incubated as specified on the upper right. Luciferase values measured with 20→20% O2 cells harvested 4 h p.t. were set to 100%. In all panels, bars represent mean values from at least three independent experiments in triplicate. Error bars indicate standard deviations.

N. Vassilaki, et al. J Virol. 2013 March;87(5):2935-2948.

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