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, 81 (15), 8122-30

Hepatitis C Virus Induces Proteolytic Cleavage of Sterol Regulatory Element Binding Proteins and Stimulates Their Phosphorylation via Oxidative Stress

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Hepatitis C Virus Induces Proteolytic Cleavage of Sterol Regulatory Element Binding Proteins and Stimulates Their Phosphorylation via Oxidative Stress

Gulam Waris et al. J Virol.

Abstract

Hepatic steatosis is a common histological feature of chronic hepatitis C. Hepatitis C virus (HCV) gene expression has been shown to alter host cell cholesterol/lipid metabolism and thus induce hepatic steatosis. Since sterol regulatory element binding proteins (SREBPs) are major regulators of lipid metabolism, we sought to determine whether genotype 2a-based HCV infection induces the expression and posttranslational activation of SREBPs. HCV infection stimulates the expression of genes related to lipogenesis. HCV induces the proteolytic cleavage of SREBPs. HCV core and NS4b derived from genotype 3a are also individually capable of inducing the proteolytic processing of SREBPs. Further, we demonstrate that HCV stimulates the phosphorylation of SREBPs. Our studies show that HCV-induced oxidative stress and subsequent activation of the phosphatidylinositol 3-kinase (PI3-K)-Akt pathway and inactivation (phosphorylation) of PTEN (phosphatase and tensin homologue) mediate the transactivation of SREBPs. HCV-induced SREBP-1 and -2 activities were sensitive to antioxidant (pyrrolidine dithiocarbamate), Ca(2+) chelator 1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-tetra(acetoxymethyl) ester (BAPTA-AM), and PI3-K inhibitor (LY294002). Collectively, these studies provide insight into the mechanisms of hepatic steatosis associated with HCV infection.

Figures

FIG. 1.
FIG. 1.
HCV induces proteolytic processing of SREBP-1 and -2. (A and B) Uninfected and HCV-infected Huh-7 cells were harvested at 48 h. Whole-cell lysates from Huh-7 and HCV-infected cells were fractionated by SDS-PAGE and immunoblotted with anti-SREBP-1/2 monoclonal antibodies. Lanes 1, Huh-7 cell lysates; lanes 2, HCV-infected cell lysates. Bottom panels represent the expression of HCV core protein as a marker of HCV infection, and actin served as an internal protein loading control. (C) Transcriptional stimulation of SREBP-1 mRNA in HCV-infected cells. Total cellular mRNA was analyzed by using SREBP-1-specific primers. Bar 1, Huh-7 cells; bar 2, HCV-infected cells. The values represent the means and standard deviations of two independent experiments performed in duplicate. (D) HCV proteins induce proteolytic processing of mature SREBP-2. Whole-cell lysates from Huh-7 cells (lanes 1, 4, and 8 and unnumbered lane between lanes 2 and 3) and cells expressing HCV nonstructural proteins, pCMV729-3010 (lane 2), HCV NS4B (1b) (lane 3), NS4B (3a) (lane 5), NS5A (lane 6), HCV core (1b) (lane 7), and core (3a) (lane 9) were fractionated by SDS-PAGE and immunoblotted with anti-SREBP-2 monoclonal antibodies. The bottom panel represents the expression of individual HCV marker proteins.
FIG. 2.
FIG. 2.
HCV induces phosphorylation of SREBP-1/2. (A and B) Huh-7 cells and HCV-infected cells were transiently transfected with N-terminus FLAG-tagged SREBP-1/2. Whole-cell lysates were immunoprecipitated with anti-FLAG monoclonal antibody, fractionated by SDS-PAGE, and immunoblotted with antiphosphoserine monoclonal antibody. Lanes 1 and 2, lysates from Huh-7 and HCV-infected cells expressing FLAG-SREBP-1 (A) and FLAG-SREBP-2 (B). Lanes 3, 4, and 5, HCV-infected cells were treated with an antioxidant, PDTC (100 μM for 6 h); a calcium chelator, BAPTA-AM (50 μM for 2 h); and a PI3-K inhibitor, LY294002 (50 μM for 12 h), respectively. The bottom panels represent the expression of N-terminus FLAG-tagged SREBP-1/2 and HCV core protein during Western blot analysis. (C) In vivo phosphorylation of SREBP-1 in HCV-infected cells. HCV-infected cells were metabolically labeled with [32P]orthophosphate (100 μCi) for 4 h. Cellular lysates were immunoprecipitated with anti-SREBP-1 antibody and subjected to SDS-PAGE followed by autoradiography. Lane 1, Huh-7 cell lysate; lane 2, HCV-infected cell lysate.
FIG. 3.
FIG. 3.
HCV proteins transactivate SREBP-2. (A) Luciferase reporter gene assays. Huh-7 cells were transfected with 500 ng of SRE-responsive SynSRE-Luc (wild type) (bar 1) and JS-15 (containing mutated SRE binding sites) luciferase plasmids along with plasmids expressing HCV proteins NS4B (1b) (bar 2), NS4B (3a) (bar 5), NS5A (bar 3), all nonstructural proteins (pCMV/729-3010) (bars 4 and 5), core (1b) (bar 7), and core (3a) (bar 6). Thirty-six hours posttransfection cellular lysates were assayed for luciferase activity. The data represent means ± standard deviations of three independent experiments performed in duplicate; the lowest level of significance was P < 0.01. The bottom panel represents the immunoblot showing the expression of individual HCV proteins as indicated by the individual expression vector used in transient transfections. 729-3010 is an expression vector that encodes all NS proteins (NS2 to NS4b) (15). NS5a expression is shown below as a representative nonstructural protein. (B) SynSRE-Luc (wild type) reporter plasmid was transfected in HCV-infected cells (black bars) as well as cotransfected with pCMV/729-3010 (white bars) and pCMV-NS4B (gray bars). Thirty-six hours posttransfection cells were treated with antioxidant (100 μM PDTC for 4 h), calcium chelator (50 μM BAPTA-AM for 2 h), and PI3-K inhibitor (50 μM LY294002 for 12 h), and cellular lysates were assayed for luciferase activity. The data represent means ± standard deviations of three independent experiments performed in duplicate; the lowest level of significance was P < 0.01.
FIG. 4.
FIG. 4.
HCV transactivates SREBP-1. (A) Luciferase reporter gene assay. Huh-7 and HCV-infected cells were transfected with 500 ng of FAS-700-Luc (wt) and FAS-mut-Luc reporter plasmids as described for Fig. 3. Results are shown as means (± standard deviations) of two independent experiments, each performed in duplicate. (B) Western blot assays. Cellular lysates from Huh-7 and HCV-infected cells were subjected to Western blot analysis using respective antibodies as indicated. Lanes 1, Huh-7 cell lysates; lanes 2, HCV-infected cell lysates. (C) Quantitative real-time PCR analysis. Total cellular mRNA was extracted, and cDNAs were prepared from Huh-7 (lane 1) and HCV-infected (lane 2) cells. Equal amounts of cDNAs were subjected to quantitative RT-PCR using LXR-specific primers. Hypoxanthine phosphoribosyltransferase (HPRT) mRNA was used as an internal control. The results represent means ± standard deviations of two independent experiments performed in triplicate. (D) Huh-7 cells and HCV-infected cells were transfected with LXR-responsive pLXRE-Luc luciferase plasmid derived from the SREBP-1c gene. Thirty-six hours posttransfection cellular lysates were prepared and assayed for luciferase activity. The data represent means ± standard deviations of three independent experiments performed in duplicate.
FIG. 5.
FIG. 5.
HCV infection induces the expression of lipogenic transcripts. Total cellular mRNA was extracted, and cDNAs were prepared from Huh-7 and HCV-infected cells. Equal amounts of cDNAs were subjected to quantitative RT-PCR using SYBR green probe. This experiment was performed in duplicates. Hypoxanthine phosphoribosyltransferase mRNA was used as a standard internal control. HMGCR, HMG-CoA reductase; Sq Synth, squalene synthase; ACL, ATP citrate lyase; SCD, stearoyl-CoA desaturase; ACC1, acetyl-CoA carboxylase.

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