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Requirement for chloride channel function during the hepatitis C virus life cycle.

Igloi Z, Mohl BP, Lippiat JD, Harris M, Mankouri J - J. Virol. (2015)

Bottom Line: Here, we show that HCV increases intracellular hepatic chloride (Cl(-)) influx that can be inhibited by selective Cl(-) channel blockers.Through pharmacological and small interfering RNA (siRNA)-mediated silencing, we demonstrate that Cl(-) channel inhibition is detrimental to HCV replication.This represents the first observation of the involvement of Cl(-) channels during the HCV life cycle.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.

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Assessment of the Cl− channel(s) required for HCV replication. (A) Expression of CLIC-1, ClC-2, ClC-3, ClC-5, and ClC-7 was ablated by transfection of 75 pmol siRNA, as demonstrated by RT-PCR using whole-cell RNA extracts from SGR–Feo–JFH-1 cells. The siRNA had minimal impact on levels of GAPDH. (B) SGR–Feo–JFH-1 cells were treated with Cl− channel siRNA or control siRNA, and luciferase expression was assessed as described for Fig. 1. Results were calculated relative to values for the transfection reagent-only controls. Error bars represent the SEM from three independent experiments. **, significant difference from the control value (P < 0.05); NS, no differences at the 0.05 significance level. (C) Huh7 cells were treated with Cl− channel siRNA or control siRNA for 48 h and infected with J6/JFH-1 RLuc virus at an MOI of 0.5 for 48 h. Cells were lysed and the levels of luciferase assayed as described for Fig. 1. Error bars represent the SEM from four independent experiments. Values are normalized to those for the transfection controls. **, significant difference from value for the untreated control (P < 0.05). NS, no differences at the 0.05 significance level.
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Figure 4: Assessment of the Cl− channel(s) required for HCV replication. (A) Expression of CLIC-1, ClC-2, ClC-3, ClC-5, and ClC-7 was ablated by transfection of 75 pmol siRNA, as demonstrated by RT-PCR using whole-cell RNA extracts from SGR–Feo–JFH-1 cells. The siRNA had minimal impact on levels of GAPDH. (B) SGR–Feo–JFH-1 cells were treated with Cl− channel siRNA or control siRNA, and luciferase expression was assessed as described for Fig. 1. Results were calculated relative to values for the transfection reagent-only controls. Error bars represent the SEM from three independent experiments. **, significant difference from the control value (P < 0.05); NS, no differences at the 0.05 significance level. (C) Huh7 cells were treated with Cl− channel siRNA or control siRNA for 48 h and infected with J6/JFH-1 RLuc virus at an MOI of 0.5 for 48 h. Cells were lysed and the levels of luciferase assayed as described for Fig. 1. Error bars represent the SEM from four independent experiments. Values are normalized to those for the transfection controls. **, significant difference from value for the untreated control (P < 0.05). NS, no differences at the 0.05 significance level.

Mentions: Given these data, we investigated the molecular identity of the Cl− channel(s) required during the HCV life cycle. To date, nearly 40 different genes that, when expressed, increase Cl− conductance have been cloned. These include the Cl− intracellular-channel (CLIC) proteins cyclic AMP (cAMP) (CFTR)-, calcium (CaCC)-, voltage-activated Cl− channels and Cl−/H+ exchangers (CLCs) as well as ligand-gated Cl− channels [GABA(A), GABA(C), and glycine]. In hepatocytes, CLIC-1, ClC-2, ClC-3, ClC-5, and ClC-7 are expressed (9). We confirmed this by reverse transcription-PCR (RT-PCR) analysis (primer sequences are available upon request) and silenced this expression through small interfering RNA (siRNA) transfection (Fig. 4A). Figure 4B shows that ClC-2, ClC-3, ClC-5, and ClC-7 silencing significantly suppressed SGR–Feo–JFH-1 replication (52% ± 6%, 31% ± 16%, 48% ± 2%, and 50% ± 10% inhibition of luciferase activity, respectively). CLIC-1 knockdown displayed no discernible effects. Since some of these CLC channels and transporters are sensitive to NPPB and IAA-94; this confirmed the importance of Cl− influx during HCV replication.


Requirement for chloride channel function during the hepatitis C virus life cycle.

Igloi Z, Mohl BP, Lippiat JD, Harris M, Mankouri J - J. Virol. (2015)

Assessment of the Cl− channel(s) required for HCV replication. (A) Expression of CLIC-1, ClC-2, ClC-3, ClC-5, and ClC-7 was ablated by transfection of 75 pmol siRNA, as demonstrated by RT-PCR using whole-cell RNA extracts from SGR–Feo–JFH-1 cells. The siRNA had minimal impact on levels of GAPDH. (B) SGR–Feo–JFH-1 cells were treated with Cl− channel siRNA or control siRNA, and luciferase expression was assessed as described for Fig. 1. Results were calculated relative to values for the transfection reagent-only controls. Error bars represent the SEM from three independent experiments. **, significant difference from the control value (P < 0.05); NS, no differences at the 0.05 significance level. (C) Huh7 cells were treated with Cl− channel siRNA or control siRNA for 48 h and infected with J6/JFH-1 RLuc virus at an MOI of 0.5 for 48 h. Cells were lysed and the levels of luciferase assayed as described for Fig. 1. Error bars represent the SEM from four independent experiments. Values are normalized to those for the transfection controls. **, significant difference from value for the untreated control (P < 0.05). NS, no differences at the 0.05 significance level.
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Figure 4: Assessment of the Cl− channel(s) required for HCV replication. (A) Expression of CLIC-1, ClC-2, ClC-3, ClC-5, and ClC-7 was ablated by transfection of 75 pmol siRNA, as demonstrated by RT-PCR using whole-cell RNA extracts from SGR–Feo–JFH-1 cells. The siRNA had minimal impact on levels of GAPDH. (B) SGR–Feo–JFH-1 cells were treated with Cl− channel siRNA or control siRNA, and luciferase expression was assessed as described for Fig. 1. Results were calculated relative to values for the transfection reagent-only controls. Error bars represent the SEM from three independent experiments. **, significant difference from the control value (P < 0.05); NS, no differences at the 0.05 significance level. (C) Huh7 cells were treated with Cl− channel siRNA or control siRNA for 48 h and infected with J6/JFH-1 RLuc virus at an MOI of 0.5 for 48 h. Cells were lysed and the levels of luciferase assayed as described for Fig. 1. Error bars represent the SEM from four independent experiments. Values are normalized to those for the transfection controls. **, significant difference from value for the untreated control (P < 0.05). NS, no differences at the 0.05 significance level.
Mentions: Given these data, we investigated the molecular identity of the Cl− channel(s) required during the HCV life cycle. To date, nearly 40 different genes that, when expressed, increase Cl− conductance have been cloned. These include the Cl− intracellular-channel (CLIC) proteins cyclic AMP (cAMP) (CFTR)-, calcium (CaCC)-, voltage-activated Cl− channels and Cl−/H+ exchangers (CLCs) as well as ligand-gated Cl− channels [GABA(A), GABA(C), and glycine]. In hepatocytes, CLIC-1, ClC-2, ClC-3, ClC-5, and ClC-7 are expressed (9). We confirmed this by reverse transcription-PCR (RT-PCR) analysis (primer sequences are available upon request) and silenced this expression through small interfering RNA (siRNA) transfection (Fig. 4A). Figure 4B shows that ClC-2, ClC-3, ClC-5, and ClC-7 silencing significantly suppressed SGR–Feo–JFH-1 replication (52% ± 6%, 31% ± 16%, 48% ± 2%, and 50% ± 10% inhibition of luciferase activity, respectively). CLIC-1 knockdown displayed no discernible effects. Since some of these CLC channels and transporters are sensitive to NPPB and IAA-94; this confirmed the importance of Cl− influx during HCV replication.

Bottom Line: Here, we show that HCV increases intracellular hepatic chloride (Cl(-)) influx that can be inhibited by selective Cl(-) channel blockers.Through pharmacological and small interfering RNA (siRNA)-mediated silencing, we demonstrate that Cl(-) channel inhibition is detrimental to HCV replication.This represents the first observation of the involvement of Cl(-) channels during the HCV life cycle.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.

Show MeSH
Related in: MedlinePlus