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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 role of Cl− channels during HCV infection. (A) Huh7 cells were pretreated with the indicated inhibitors for 45 min and infected with J6/JFH-1 RLuc virus at an MOI of 0.5 in the presence of each compound for 48 h. Cells were lysed and the levels of luciferase assayed as described for Fig. 1. (B) JFH-1 supernatants were produced by electroporation of cells with JFH-1 RNA and collection of the media from repeated passages for 2 weeks. Virus inoculum was clarified by centrifugation and added to Huh7 cells (5 × 104 cells in six-well plates) in the presence of the indicated compounds. Cells were washed 24 h postinfection and were treated with medium plus compound for a further 48 h. Virus supernatants were collected and titrated onto Huh7 cells to assess numbers of focus-forming units (FFU)/ml. (C) Sample Western blots from the experiment discussed in panel B immunoblotted for NS5A, core, and GAPDH (loading control) are shown. (D) Experiments were performed as described for panel B in the presence of KCl and TEA at concentrations assessed in the experiment illustrated in Fig. 1. (E) Huh7 cells were infected with JFH-1 supernatants for 24 h. Cells were washed and replaced with medium plus compound for a further 48 h. Virus supernatants were collected, and virus production was assessed by focus-forming assays as described for panel B. (F) Compounds were mixed with virus inoculum in DMEM for 1 h and Huh7 cells infected at 37°C for 3 h. Cells were washed three times in phosphate-buffered saline (PBS) to remove unbound virus, and virus production was assessed 48 postinfection by focus-forming assays of virus supernatants. AP33 was included in these assays to inhibit HCV entry (50 mg/ml). All results were calculated relative to values for the untreated controls. **, significant difference from control value (P < 0.05); NS, no differences at the 0.05 significance level.
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Figure 3: Assessment of the role of Cl− channels during HCV infection. (A) Huh7 cells were pretreated with the indicated inhibitors for 45 min and infected with J6/JFH-1 RLuc virus at an MOI of 0.5 in the presence of each compound for 48 h. Cells were lysed and the levels of luciferase assayed as described for Fig. 1. (B) JFH-1 supernatants were produced by electroporation of cells with JFH-1 RNA and collection of the media from repeated passages for 2 weeks. Virus inoculum was clarified by centrifugation and added to Huh7 cells (5 × 104 cells in six-well plates) in the presence of the indicated compounds. Cells were washed 24 h postinfection and were treated with medium plus compound for a further 48 h. Virus supernatants were collected and titrated onto Huh7 cells to assess numbers of focus-forming units (FFU)/ml. (C) Sample Western blots from the experiment discussed in panel B immunoblotted for NS5A, core, and GAPDH (loading control) are shown. (D) Experiments were performed as described for panel B in the presence of KCl and TEA at concentrations assessed in the experiment illustrated in Fig. 1. (E) Huh7 cells were infected with JFH-1 supernatants for 24 h. Cells were washed and replaced with medium plus compound for a further 48 h. Virus supernatants were collected, and virus production was assessed by focus-forming assays as described for panel B. (F) Compounds were mixed with virus inoculum in DMEM for 1 h and Huh7 cells infected at 37°C for 3 h. Cells were washed three times in phosphate-buffered saline (PBS) to remove unbound virus, and virus production was assessed 48 postinfection by focus-forming assays of virus supernatants. AP33 was included in these assays to inhibit HCV entry (50 mg/ml). All results were calculated relative to values for the untreated controls. **, significant difference from control value (P < 0.05); NS, no differences at the 0.05 significance level.

Mentions: Since SGR–Feo–JFH-1-harboring cells express only the HCV nonstructural proteins NS3 to NS5B, it was important to determine whether Cl− channel inhibition suppressed HCV replication in the context of virus-infected cells (23). We initially used a monocistronic full-length HCV chimeric genotype 2a virus, J6/JFH1, which is fully infectious in cell culture and expresses Renilla luciferase, herein termed J6/JFH-1 RLuc (24). Assays were performed by virus infection (multiplicity of infection [MOI] of 0.5) in the presence of each channel modulator, and luciferase expression was analyzed 48 h postinfection (p.i.). Figure 3A shows that NPPB and IAA-94 treatment significantly decreased J6/JFH-1 RLuc activity (67% ± 20% and 63% ± 5% inhibition, respectively) confirming a dependence on Cl− influx during the virus life cycle. When these assays were performed in the presence of DIDS (100 μM), J6/JFH-1 RLuc activity also decreased by 77% ± 4% at concentrations that did not affect SGR–Feo–JFH-1 replication (Fig. 3A). To verify these data, we directly infected Huh7 cells with full-length JFH-1 virus (25) in the presence of each Cl− inhibitor and measured the production of infectious virions by focus-forming assay. As shown in Fig. 3B, virus yields were significantly lower in IAA-94-, NPPB-, and DIDS-treated cells (87% ± 14%, 81% ± 23%, and 72% ± 22% inhibition, respectively). This was paralleled by a decrease of both NS5A and core protein expression in virus lysates as assessed by Western blot analysis (Fig. 3C). No effects on JFH-1 virus production were observed when TEA or KCl was assessed in these assays (Fig. 3D). We subsequently performed time-of-addition focus reduction assays using JFH-1 virus inoculum to assess the effects of DIDS over the time course of HCV infection. Cells were treated with each inhibitor 24 h p.i., and virus production was assessed 72 h p.i. Figure 3E shows that DCV, NPPB, and IAA-94 reduced JFH-1 virus production when added postinfection (92% ± 9%, 81% ± 23%, and 72% ± 22% inhibition, respectively), consistent with a block of HCV replication. DIDS however, failed to reduce virus production relative to that in the untreated wells, consistent with a lack of inhibition of HCV replication. To further determine which steps of the HCV life cycle are impaired by DIDS, we examined the effects of each Cl− channel inhibitor on virus entry by adding them to JFH-1 inoculum during the initial 3 h of virus infection (26). The HCV-neutralizing mouse monoclonal E2 antibody AP33, a characterized inhibitor of HCV entry, was included in these assays for verification (27). Figure 3F shows that, while AP33 (50 μg/ml) inhibited HCV entry by 72% ± 11%, IAA-94, NPPB, and DIDS did not impede viral entry. These observations suggest that a DIDS-sensitive Cl− channel can inhibit early postentry virion trafficking and/or early replication events but does not inhibit virus entry or replication following the establishment of infection.


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 role of Cl− channels during HCV infection. (A) Huh7 cells were pretreated with the indicated inhibitors for 45 min and infected with J6/JFH-1 RLuc virus at an MOI of 0.5 in the presence of each compound for 48 h. Cells were lysed and the levels of luciferase assayed as described for Fig. 1. (B) JFH-1 supernatants were produced by electroporation of cells with JFH-1 RNA and collection of the media from repeated passages for 2 weeks. Virus inoculum was clarified by centrifugation and added to Huh7 cells (5 × 104 cells in six-well plates) in the presence of the indicated compounds. Cells were washed 24 h postinfection and were treated with medium plus compound for a further 48 h. Virus supernatants were collected and titrated onto Huh7 cells to assess numbers of focus-forming units (FFU)/ml. (C) Sample Western blots from the experiment discussed in panel B immunoblotted for NS5A, core, and GAPDH (loading control) are shown. (D) Experiments were performed as described for panel B in the presence of KCl and TEA at concentrations assessed in the experiment illustrated in Fig. 1. (E) Huh7 cells were infected with JFH-1 supernatants for 24 h. Cells were washed and replaced with medium plus compound for a further 48 h. Virus supernatants were collected, and virus production was assessed by focus-forming assays as described for panel B. (F) Compounds were mixed with virus inoculum in DMEM for 1 h and Huh7 cells infected at 37°C for 3 h. Cells were washed three times in phosphate-buffered saline (PBS) to remove unbound virus, and virus production was assessed 48 postinfection by focus-forming assays of virus supernatants. AP33 was included in these assays to inhibit HCV entry (50 mg/ml). All results were calculated relative to values for the untreated controls. **, significant difference from control value (P < 0.05); NS, no differences at the 0.05 significance level.
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Figure 3: Assessment of the role of Cl− channels during HCV infection. (A) Huh7 cells were pretreated with the indicated inhibitors for 45 min and infected with J6/JFH-1 RLuc virus at an MOI of 0.5 in the presence of each compound for 48 h. Cells were lysed and the levels of luciferase assayed as described for Fig. 1. (B) JFH-1 supernatants were produced by electroporation of cells with JFH-1 RNA and collection of the media from repeated passages for 2 weeks. Virus inoculum was clarified by centrifugation and added to Huh7 cells (5 × 104 cells in six-well plates) in the presence of the indicated compounds. Cells were washed 24 h postinfection and were treated with medium plus compound for a further 48 h. Virus supernatants were collected and titrated onto Huh7 cells to assess numbers of focus-forming units (FFU)/ml. (C) Sample Western blots from the experiment discussed in panel B immunoblotted for NS5A, core, and GAPDH (loading control) are shown. (D) Experiments were performed as described for panel B in the presence of KCl and TEA at concentrations assessed in the experiment illustrated in Fig. 1. (E) Huh7 cells were infected with JFH-1 supernatants for 24 h. Cells were washed and replaced with medium plus compound for a further 48 h. Virus supernatants were collected, and virus production was assessed by focus-forming assays as described for panel B. (F) Compounds were mixed with virus inoculum in DMEM for 1 h and Huh7 cells infected at 37°C for 3 h. Cells were washed three times in phosphate-buffered saline (PBS) to remove unbound virus, and virus production was assessed 48 postinfection by focus-forming assays of virus supernatants. AP33 was included in these assays to inhibit HCV entry (50 mg/ml). All results were calculated relative to values for the untreated controls. **, significant difference from control value (P < 0.05); NS, no differences at the 0.05 significance level.
Mentions: Since SGR–Feo–JFH-1-harboring cells express only the HCV nonstructural proteins NS3 to NS5B, it was important to determine whether Cl− channel inhibition suppressed HCV replication in the context of virus-infected cells (23). We initially used a monocistronic full-length HCV chimeric genotype 2a virus, J6/JFH1, which is fully infectious in cell culture and expresses Renilla luciferase, herein termed J6/JFH-1 RLuc (24). Assays were performed by virus infection (multiplicity of infection [MOI] of 0.5) in the presence of each channel modulator, and luciferase expression was analyzed 48 h postinfection (p.i.). Figure 3A shows that NPPB and IAA-94 treatment significantly decreased J6/JFH-1 RLuc activity (67% ± 20% and 63% ± 5% inhibition, respectively) confirming a dependence on Cl− influx during the virus life cycle. When these assays were performed in the presence of DIDS (100 μM), J6/JFH-1 RLuc activity also decreased by 77% ± 4% at concentrations that did not affect SGR–Feo–JFH-1 replication (Fig. 3A). To verify these data, we directly infected Huh7 cells with full-length JFH-1 virus (25) in the presence of each Cl− inhibitor and measured the production of infectious virions by focus-forming assay. As shown in Fig. 3B, virus yields were significantly lower in IAA-94-, NPPB-, and DIDS-treated cells (87% ± 14%, 81% ± 23%, and 72% ± 22% inhibition, respectively). This was paralleled by a decrease of both NS5A and core protein expression in virus lysates as assessed by Western blot analysis (Fig. 3C). No effects on JFH-1 virus production were observed when TEA or KCl was assessed in these assays (Fig. 3D). We subsequently performed time-of-addition focus reduction assays using JFH-1 virus inoculum to assess the effects of DIDS over the time course of HCV infection. Cells were treated with each inhibitor 24 h p.i., and virus production was assessed 72 h p.i. Figure 3E shows that DCV, NPPB, and IAA-94 reduced JFH-1 virus production when added postinfection (92% ± 9%, 81% ± 23%, and 72% ± 22% inhibition, respectively), consistent with a block of HCV replication. DIDS however, failed to reduce virus production relative to that in the untreated wells, consistent with a lack of inhibition of HCV replication. To further determine which steps of the HCV life cycle are impaired by DIDS, we examined the effects of each Cl− channel inhibitor on virus entry by adding them to JFH-1 inoculum during the initial 3 h of virus infection (26). The HCV-neutralizing mouse monoclonal E2 antibody AP33, a characterized inhibitor of HCV entry, was included in these assays for verification (27). Figure 3F shows that, while AP33 (50 μg/ml) inhibited HCV entry by 72% ± 11%, IAA-94, NPPB, and DIDS did not impede viral entry. These observations suggest that a DIDS-sensitive Cl− channel can inhibit early postentry virion trafficking and/or early replication events but does not inhibit virus entry or replication following the establishment of infection.

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