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Hepatitis C virus attenuates mitochondrial lipid β-oxidation by downregulating mitochondrial trifunctional-protein expression.

Amako Y, Munakata T, Kohara M, Siddiqui A, Peers C, Harris M - J. Virol. (2015)

Bottom Line: However, little is known about the effects of HCV on lipid β-oxidation.Here we show that in HCV-infected Huh7.5 cells, lipid β-oxidation was significantly attenuated.This study revealed that HCV impairs mitochondrial lipid β-oxidation, which results in low lipid combustion.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom amako-yt@igakuken.or.jp m.harris@leeds.ac.uk.

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MTP protein depletion leads to suppression of the type I IFN response. MTP depletion does not directly affect HCV replication but suppresses the type I IFN response. (A) Huh7.5 cells were transduced with the indicated lentiviral shRNA vectors, and after 24 h of incubation, cells were infected with HCV Jc1-p7NLuc2A (MOI = 0.05 FFU/cell) (Fig. 5). Infected cells were lysed at days 2 and 4 after HCV infection and assayed by the nanoluciferase A Glo assay to assess HCV replication. (B) MTP depletion renders host cells less responsive to exogenous IFN-α to suppress HCV replication. Huh7.5 cells were transduced with the indicated lentiviral shRNA vectors. Two days later, cells were subsequently infected with HCV (MOI = 0.05 FFU/cell), incubated for 4 days, and plated in a 96-well plate at 1 × 104 cells per well for IFN-α treatment for a further 2 days. The levels of viral replication were assayed using Nano-Glo luciferase assay reagent as per the manufacturer's instruction (n = 6). (C) MTP depletion reduces the ISG response. Lentiviral-vector-mediated shRNAs as indicated were used to downregulate MTP gene expression. Transduced cells were transfected with pISRE-Luc and pRL-TK and then stimulated with IFN-α at the indicated concentrations for 5 h prior to analysis by the dual-luciferase reporter assay (n = 4). Error bars represent SEM.
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Figure 6: MTP protein depletion leads to suppression of the type I IFN response. MTP depletion does not directly affect HCV replication but suppresses the type I IFN response. (A) Huh7.5 cells were transduced with the indicated lentiviral shRNA vectors, and after 24 h of incubation, cells were infected with HCV Jc1-p7NLuc2A (MOI = 0.05 FFU/cell) (Fig. 5). Infected cells were lysed at days 2 and 4 after HCV infection and assayed by the nanoluciferase A Glo assay to assess HCV replication. (B) MTP depletion renders host cells less responsive to exogenous IFN-α to suppress HCV replication. Huh7.5 cells were transduced with the indicated lentiviral shRNA vectors. Two days later, cells were subsequently infected with HCV (MOI = 0.05 FFU/cell), incubated for 4 days, and plated in a 96-well plate at 1 × 104 cells per well for IFN-α treatment for a further 2 days. The levels of viral replication were assayed using Nano-Glo luciferase assay reagent as per the manufacturer's instruction (n = 6). (C) MTP depletion reduces the ISG response. Lentiviral-vector-mediated shRNAs as indicated were used to downregulate MTP gene expression. Transduced cells were transfected with pISRE-Luc and pRL-TK and then stimulated with IFN-α at the indicated concentrations for 5 h prior to analysis by the dual-luciferase reporter assay (n = 4). Error bars represent SEM.

Mentions: Plasmids pISRE-Luc and pRL-TK (20:1, wt/wt, ratio) were transfected into Huh7.5 cells on a 12-well plate with Fugene 6 transfection reagent. Transfected cells were split into a 96-well plate at 24 h posttransfection. Cells were further incubated for 48 h, and during the last 5 h, they were exposed to IFN-α at the concentrations indicated in Fig. 6. Measurement of luciferase activity was performed using the dual-luciferase reporter assay system for pISRE-Luc and pRL-TK.


Hepatitis C virus attenuates mitochondrial lipid β-oxidation by downregulating mitochondrial trifunctional-protein expression.

Amako Y, Munakata T, Kohara M, Siddiqui A, Peers C, Harris M - J. Virol. (2015)

MTP protein depletion leads to suppression of the type I IFN response. MTP depletion does not directly affect HCV replication but suppresses the type I IFN response. (A) Huh7.5 cells were transduced with the indicated lentiviral shRNA vectors, and after 24 h of incubation, cells were infected with HCV Jc1-p7NLuc2A (MOI = 0.05 FFU/cell) (Fig. 5). Infected cells were lysed at days 2 and 4 after HCV infection and assayed by the nanoluciferase A Glo assay to assess HCV replication. (B) MTP depletion renders host cells less responsive to exogenous IFN-α to suppress HCV replication. Huh7.5 cells were transduced with the indicated lentiviral shRNA vectors. Two days later, cells were subsequently infected with HCV (MOI = 0.05 FFU/cell), incubated for 4 days, and plated in a 96-well plate at 1 × 104 cells per well for IFN-α treatment for a further 2 days. The levels of viral replication were assayed using Nano-Glo luciferase assay reagent as per the manufacturer's instruction (n = 6). (C) MTP depletion reduces the ISG response. Lentiviral-vector-mediated shRNAs as indicated were used to downregulate MTP gene expression. Transduced cells were transfected with pISRE-Luc and pRL-TK and then stimulated with IFN-α at the indicated concentrations for 5 h prior to analysis by the dual-luciferase reporter assay (n = 4). Error bars represent SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4442397&req=5

Figure 6: MTP protein depletion leads to suppression of the type I IFN response. MTP depletion does not directly affect HCV replication but suppresses the type I IFN response. (A) Huh7.5 cells were transduced with the indicated lentiviral shRNA vectors, and after 24 h of incubation, cells were infected with HCV Jc1-p7NLuc2A (MOI = 0.05 FFU/cell) (Fig. 5). Infected cells were lysed at days 2 and 4 after HCV infection and assayed by the nanoluciferase A Glo assay to assess HCV replication. (B) MTP depletion renders host cells less responsive to exogenous IFN-α to suppress HCV replication. Huh7.5 cells were transduced with the indicated lentiviral shRNA vectors. Two days later, cells were subsequently infected with HCV (MOI = 0.05 FFU/cell), incubated for 4 days, and plated in a 96-well plate at 1 × 104 cells per well for IFN-α treatment for a further 2 days. The levels of viral replication were assayed using Nano-Glo luciferase assay reagent as per the manufacturer's instruction (n = 6). (C) MTP depletion reduces the ISG response. Lentiviral-vector-mediated shRNAs as indicated were used to downregulate MTP gene expression. Transduced cells were transfected with pISRE-Luc and pRL-TK and then stimulated with IFN-α at the indicated concentrations for 5 h prior to analysis by the dual-luciferase reporter assay (n = 4). Error bars represent SEM.
Mentions: Plasmids pISRE-Luc and pRL-TK (20:1, wt/wt, ratio) were transfected into Huh7.5 cells on a 12-well plate with Fugene 6 transfection reagent. Transfected cells were split into a 96-well plate at 24 h posttransfection. Cells were further incubated for 48 h, and during the last 5 h, they were exposed to IFN-α at the concentrations indicated in Fig. 6. Measurement of luciferase activity was performed using the dual-luciferase reporter assay system for pISRE-Luc and pRL-TK.

Bottom Line: However, little is known about the effects of HCV on lipid β-oxidation.Here we show that in HCV-infected Huh7.5 cells, lipid β-oxidation was significantly attenuated.This study revealed that HCV impairs mitochondrial lipid β-oxidation, which results in low lipid combustion.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom amako-yt@igakuken.or.jp m.harris@leeds.ac.uk.

Show MeSH
Related in: MedlinePlus