The Roles of Endoplasmic Reticulum Overload Response Induced by HCV and NS4B Protein in Human Hepatocyte Viability and Virus Replication

<div><p>Hepatitis C virus (HCV) replication is associated with endoplasmic reticulum (ER) and its infection triggers ER stress. In response to ER stress, ER overload response (EOR) can be activated, which involves the release of Ca²⁺ from ER, production of reactive oxygen species (ROS) and activation of nuclear factor κB (NF-κB). We have previously reported that HCV NS4B expression activates NF-κB via EOR-Ca²⁺-ROS pathway. Here, we showed that NS4B expression and HCV infection activated cancer-related NF-κB signaling pathway and induced the expression of cancer-related NF-κB target genes via EOR-Ca²⁺-ROS pathway. Moreover, we found that HCV-activated EOR-Ca²⁺-ROS pathway had profound effects on host cell viability and HCV replication. HCV infection induced human hepatocyte death by EOR-Ca²⁺-ROS pathway, whereas activation of EOR-Ca²⁺-ROS-NF-κB pathway increased the cell viability. Meanwhile, EOR-Ca²⁺-ROS-NF-κB pathway inhibited acute HCV replication, which could alleviate the detrimental effect of HCV on cell viability and enhance chronic HCV infection. Together, our findings provide new insights into the functions of EOR-Ca²⁺-ROS-NF-κB pathway in natural HCV replication and pathogenesis.</p></div

The effect of HCV on human hepatocyte viability via EOR.

<p>(A). Huh7.5.1 cells were infected with JFH1 at a virus titer (IU/cell) of 0, 0.02, 1 and 5. At 48 h postinfection, cell viability was assessed by using Cell Titre-Glo assay. Values are means ± SD (n = 3). * P < 0.05. (B). Huh7.5.1 cells were infected with JFH1 at a virus titer (IU/cell) of 5 and treated with TMB-8 (100 μM) for 4 h, NAC (30 mM) for 8 h and SN50 (40 μM) for 4 h as indicated. Mock-infected Huh7.5.1 cells were used as controls. At 48 h postinfection, cell viability was assessed by using Cell Titre-Glo assay. Values are means ± SD (n = 3). * P < 0.05. (C). Primary human hepatocytes in 96-well plates were infected with JFH1 at a virus titer (IU/cell) of 1 and treated with TMB-8 (100 μM) for 4 h, NAC (30 mM) for 8 h and SN50 (40 μM) for 4 h as indicated. Mock-infected primary human hepatocytes were used as controls. At 48 h postinfectiom, cell viability was assessed using Cell Titre-Glo assay. Values are means ± SD (n = 3). * P < 0.05.</p

NS4B activates cancer-related NF-κB signaling pathway via EOR in human hepatocytes.

<p>Huh-7 cells stably expressing NS4B and control cells were treated with NF-κB inhibitor SN50 (40 μM) for 4 h or untreated as indicated. To show this effect is specific to NS4B, Huh-7 cells that stably expressed NS4B were treated with NS4B siRNA or control siRNA at a final concentration of 25 nM. (A). Real-time RT-PCR analysis of NS4B, C-myc, Mcl-1, Cyclin D1, and MMP-9. GAPDH acts as internal control. Values are means ± SD (n = 3). * P < 0.05. (B). Western blot analysis of the protein levels of C-myc, Mcl-1, Cyclin D1, MMP-9, and NS4B. Actin protein was used as internal controls. (C) and (D). Huh-7 cells stably expressing NS4B were co-transfected with plasmids pNF-κB-Luc and pRL-CMV, and treated with TMB-8 (100 μM) for 4 h and NAC (30 mM) for 8 h as indicated. At 48 h posttransfection, cells were subjected to luciferase assay for NF-κB activation (C) and real-time RT-PCR for analyzing transcripts of C-myc, Cyclin D1, Mcl-1 and MMP-9 (D). Values are means ± SD (n = 3). * P < 0.05. E. Huh-7 cells stably expressing NS4B were treated with TMB-8 (100 μM) for 4 h and NAC (30 mM) for 8 h as indicated. The protein levels of C-myc, Mcl-1, Cyclin D1, HCV NS4B, MMP-9 and phospho-IκBα were analyzed by western blot with indicated antibodies. Phospho-IκBα was probed to confirm NF-κB activation.</p

The effect of NS4B on human hepatocyte viability via EOR.

<p>Huh7 cells stably expressing NS4B or control cells were plated on 24-well plates and treated with SN50 (40 μM) for 4 h, NAC (30 mM) for 8 h and TMB-8 (100 μM) for 4 h as indicated. After 48 h, cell viability was assessed using Cell Titre-Glo (A) and WST (B) assays. Values are means ± SD (n = 3). * P < 0.05.</p

Activation of cancer-related NF-κB signaling pathway by HCV via EOR in human hepatoma cells.

<p>(A). Huh7.5.1 cells were infected with JFH1 at a virus titer (IU/cell) of 0, 0.02, 1 and 5. At 48 h postinfection, cells were subjected to indirect immunofluorescece with mouse anti-Core antibody and Alexa FluorR 555 anti-mouse secondary antibody. Nuclei were stained with DAPI. (B). Huh7.5.1 cells were infected with JFH1 at a virus titer (IU/cell) of 0, 0.02, 1 and 5, and transfected with plasmids consisting of NF-κB-Luc and pRL-CMV. After 48 h, cells were subjected to luciferase assay for NF-κB activation. Values are means ± SD (n = 3). * P < 0.05. Scale bars represent 50 μm. (C) and (D). Huh7.5.1 cells were infected with JFH1 at a virus titer (IU/cell) of 5, and treated with SN50 (40 μM) for 4 h, TMB-8 (100 μM) for 4 h and NAC (30 mM) for 8 h as indicated. (C). Western blot analysis of protein levels of C-myc, Mcl-1, Cyclin D1, MMP-9, phospho-IκBα, NS4B and actin in cells at 48 h posttransfection. Actin protein bands act as internal control. p50 protein accumulation in the nuclear extracts was also analyzed by western blot. YY1 acts as a nuclear-specific control. (D). Real-time RT-PCR analysis of C-myc, Mcl-1, Cyclin D1 and MMP-9. GAPDH act as internal control. Values are means ± SD (n = 3). * P < 0.05. (E). Primary human hepatocytes in 24-well plate were infected with JFH1 at a virus titer (IU/cell) of 1. Mock-infected primary human hepatocytes were used as controls. At 48 h postinfection, Core, NS4B and actin proteins were determined by Western blot. (F). Primary human hepatocytes in 96-well plates were infected with JFH1 at a virus titer (IU/cell) of 1, and treated with SN50 (40 μM) for 4 h, TMB-8 (100 μM) for 4 h and NAC (30 mM) for 8 h as indicated. At 48 h postinfection, transcripts of C-myc, Mcl-1, Cyclin D1 and MMP-9 in cells were analyzed by real-time RT-PCR. GAPDH acts as internal control. Values are means ± SD (n = 3). * P < 0.05. (G). Mock-infected primary human hepatocytes in 96-well plates and treated with SN50 (40 μM) for 4 h, TMB-8 (100 μM) for 4 h and NAC (30 mM) for 8 h as indicated. At 48 h postinfection, transcripts of C-myc, Mcl-1, Cyclin D1 and MMP-9 in cells were analyzed by real-time RT-PCR. GAPDH acts as internal control. Values are means ± SD (n = 3). * P < 0.05. (H). Huh7.5.1 cells were infected with JFH1 at a virus titer (IU/cell) of 5, transfected with plasmids consisting of NF-κB-Luc and pRL-CMV, and treated with SN50 (40 μM) for 4 h, Ryanodine (100 nM) for 4 h, Ruthenium red (50 μM) for 4 h and NAC (30 mM) for 8 h as indicated. At 48 h posttranfection, cells were subjected to luciferase assay. Values are means ± SD (n = 3). * P < 0.05.</p