Hepatitis B virus X protein stimulates the mitochondrial translocation of Raf-1 via oxidative stress - PubMed (original) (raw)
Hepatitis B virus X protein stimulates the mitochondrial translocation of Raf-1 via oxidative stress
Jun Chen et al. J Virol. 2007 Jun.
Abstract
The human hepatitis B virus (HBV) X protein (HBx) plays a crucial role(s) in the viral life cycle and contributes to the onset of hepatocellular carcinoma (HCC). HBx caused the mitochondrial translocation of Raf-1 kinase either alone or in the context of whole-viral-genome transfections. Mitochondrial translocation of Raf-1 is mediated by HBx-induced oxidative stress and was dependent upon the phosphorylation of Raf-1 at the serine338/339 and Y340/341 residues by p21-activated protein kinase 1 and Src kinase, respectively. These studies provide an insight into the mechanisms by which HBV induces intracellular events relevant to liver disease pathogenesis, including HCC.
Figures
FIG. 1.
HBx protein induces Raf-1 mitochondrial translocation. (A) Raf-1 levels in untransfected and pCMVXF-transfected Huh-7 cellular lysates. Western blot analysis was carried out using anti-Raf-1 kinase antibody. Anti-actin was used as a protein loading control. (B) Mitochondrial preparations from untransfected and pCMVXF-transfected Huh-7 cells were used for Western blot analysis using anti-Raf-1 kinase antibody. VDAC serves as a mitochondrial marker. Anti-Flag was used to monitor HBx expression, and anti-LDH was used to monitor for cytoplasmic contamination. (C) Cytoplasmic fractions from untransfected and pCMVXF-transfected Huh-7 cells were analyzed by Western blot assays using anti-Raf-1, anti-Flag (which detects HBx), and anti-LDH. LDH is used here as a cytoplasmic marker. (D) Mitochondria were fractionated (9) from untransfected Huh-7 cells and cells transfected with whole-HBV-genome plasmids (HBV1.3L) and an X-defective mutant plasmid of the HBV genome [HBV1.3L (ΔX)] (gift from J. Ou, USC). Western blot analysis was carried out using anti-Raf-1 kinase antibody and anti-electron transport factor (anti-ETF; a mitochondrial marker) antibody. HBx protein expression was monitored by first immunoprecipitating with anti-HBx antibody (16), followed by immunoblotting with the same antiserum (16). (E) Raf-1 mitochondrial translocation (9) in the HBx-transgenic mouse. Mitochondria were fractionated from normal and HBx-transgenic mice (gift from James Ou). HBx was expressed under its native promoter/enhancer (16). Western blot analysis was performed on the mitochondrial preparation. Lane 1, normal mouse liver tissue; lane 2, HBx-transgenic mouse liver Western blots using anti-Raf-1. ETF is used as a mitochondrial marker. HBx expression in the HBx-transgenic mice was determined using anti-HBx antibody by immunoprecipitation (IP), followed by immunoblot analysis with the same antibody (gift from Betty Slagle) (16).
FIG. 2.
HBx protein activates both Src-dependent tyrosine and PAK-1-dependent serine phosphorylation of Raf-1 and causes its mitochondrial translocation. (A) HBx activates serine phosphorylation of mitochondrial Raf-1. Western blot analysis was performed on isolated mitochondria fractionated from untransfected Huh-7 cells and those transiently transfected with the pCMV4X plasmid using the anti-Raf-1 Ser338 monoclonal antibody, which recognizes serine338-phosphorylated Raf-1 kinase (Upstate Biotechnologies). Anti-electron transport factor (anti-ETF) was used to detect ETF, a mitochondrial marker. HBx was monitored using anti-Flag antibody. (B) Pak-1 is required for Raf-1 translocation. Results are shown for Western blot analysis of isolated mitochondria prepared from untransfected Huh-7 cells (lane 1), Huh-7 cells transiently transfected with the pCMV4X plasmid (lane 2), Huh-7 cells cotransfected with pCMV4X-Flag (HBx), and dominant-negative Pak-1 protein (the PAK-1 inhibitory domain containing PAK83-149) (lane 2) using anti-Raf-1 antibody. ETF serves as a mitochondrial marker. HBx expression was monitored using anti-Flag antibody (an internal control). (C) Western blot analysis was carried out as described for panel B, except that a Src dominant-negative mutant (gift from R. Jove) was used for cotransfection along with the pCMV4X (HBx) plasmid (lane 2).
FIG. 3.
HBx induces Raf-1 mitochondrial translocation via oxidative stress. Western blot analysis of isolated mitochondria prepared from untransfected Huh-7 cells (lane 4) and Huh-7 cells transiently transfected with pCMV4X (lanes 1 to 3) is shown. Cells were either untreated (lanes 1 and 4) or treated with antioxidants pyrrolidine dithiocarbamate (PDTC; 50 μM) (lane 2) and _N_-acetyl cysteine (NAC; 30 mM) (lane 3) for 6 h each and subjected to Western blot assays using Raf-1 antibody. Electron transport factor (ETF) serves as a mitochondrial marker. Anti-Flag was used to detect HBx (internal control).
FIG. 4.
HBx protein directly binds mitochondrially associated Raf-1. (A) Mitochondrial preparations from untransfected and pCMV4X-transfected Huh-7 cells were first immunoprecipitated with anti-Raf-1, followed by immunoblotting with anti-Flag (which detects HBx) (lane 1) antibodies. (B and C) Western blot analysis of the mitochondrial preparations with anti-Flag (HBx) and anti-electron transport factor (anti-ETF) (a mitochondrial marker).
References
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- Bouchard, M. J., L. H. Wang, and R. J. Schneider. 2001. Calcium signaling by HBx protein in hepatitis B virus DNA replication. Science 294:2376-2378. - PubMed
- Cougot, D., C. Neuveut, and M. A. Buendia. 2005. HBV-induced carcinogenesis. J. Clin. Virol. 34(Suppl. 1):875-878. - PubMed
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