Hepatitis B virus x protein induces perinuclear mitochondrial clustering in microtubule- and Dynein-dependent manners - PubMed (original) (raw)
Hepatitis B virus x protein induces perinuclear mitochondrial clustering in microtubule- and Dynein-dependent manners
Sujeong Kim et al. J Virol. 2007 Feb.
Abstract
The hepatitis B virus (HBV) X protein (HBx) is thought to play a key role in HBV replication and the development of liver cancer. It became apparent that HBx induces mitochondrial clustering at the nuclear periphery, but the molecular basis for mitochondrial clustering is not understood. Since mitochondria move along the cytoskeleton as a cargo of motor proteins, we hypothesized that mitochondrial clustering induced by HBx occurs by an altered intracellular motility. Here, we demonstrated that the treatment of HBx-expressing cells with a microtubule-disrupting drug (nocodazole) abrogated mitochondrial clustering, while the removal of nocodazole restored clustering within 30 to 60 min, indicating that mitochondrial transport is occurring in a microtubule-dependent manner. The addition of a cytochalasin D-disrupting actin filament, however, did not measurably affect mitochondrial clustering. Mitochondrial clustering was further studied by observations of HBV-related hepatoma cells and HBV-replicating cells. Importantly, the abrogation of the dynein activity in HBx-expressing cells by microinjection of a neutralizing anti-dynein intermediate-chain antibody, dynamitin overexpression, or the addition of a dynein ATPase inhibitor significantly suppressed the mitochondrial clustering. In addition, HBx induced the activation of the p38 mitogen-activated protein kinase (MAPK) and inhibition of the p38 kinase activity by SB203580-attenuated HBx-induced mitochondrial clustering. Taken together, HBx activation of the p38 MAPK contributed to the increase in the microtubule-dependent dynein activity. The data suggest that HBx plays a novel regulatory role in subcellular transport systems, perhaps facilitating the process of maturation and/or assembly of progeny particles during HBV replication. Furthermore, mitochondrion aggregation induced by HBx may represent a cellular process that underlies disease progression during chronic viral infection.
Figures
FIG. 1.
HBx induces perinuclear clustering of mitochondria in HBx-expressing Chang cells. (A) Parental Chang, control vector-transfected ChangV9, and HBx-expressing ChangX-31 and ChangX-34 cells were incubated for 30 min with MitoTrackerRed. Cells were fixed with cold methanol-acetone (1:1) and examined for subcellular mitochondrial distribution under a fluorescence microscope. The microscopic pictures are representative of six independent experiments. The crescent pattern of mitochondrion distribution was used as a criterion to judge mitochondrial clustering. (B) Electron microscopic view of subcellular organelles in Chang and ChangX-34 cells. Asterisks mark areas that are free of organelles. The right panel shows an enlargement of the inset. (C) Expression levels of the HBx protein were determined by Western blotting using rabbit anti-HBx antibody. α-Tubulin levels were used as a loading control in each lane.
FIG. 2.
Mitochondria migrate toward the MTOC in a microtubule-dependent manner in HBx-expressing cells. (A) Subcellular localizations of the HBx protein and mitochondria were visualized by immunofluorescence staining using anti-HBx antibody (green) and MitoTrackerRed (red) under a fluorescence microscope. The overlaid image was shown in the right panel, and the scale bar indicates 10 μm. (B) Chang and ChangX-34 cells were incubated with MitoTrackerRed for 30 min, and nuclei were subsequently stained with DAPI. The MTOC in ChangX-34 cells was identified by anti-pericentrin immunofluorescence (right panel). (C) Microtubules were depolymerized by the addition of 3.3 μM nocodazole (Noc) for 2 h and repolymerized by the removal of nocodazole by several washes with PBS. Mitochondria and α-tubulin (green) are representative of four independent experiments. (D) Actins were depolymerized by treatment with 1 μM cytochalasin D (Cyt. D) for 1 h and repolymerized by the removal of cytochalasin D. Mitochondria and β-actin (green) staining are representative of three different experiments. The crescent pattern of mitochondrion distribution was used as the criterion for judging mitochondrial clustering.
FIG. 2.
Mitochondria migrate toward the MTOC in a microtubule-dependent manner in HBx-expressing cells. (A) Subcellular localizations of the HBx protein and mitochondria were visualized by immunofluorescence staining using anti-HBx antibody (green) and MitoTrackerRed (red) under a fluorescence microscope. The overlaid image was shown in the right panel, and the scale bar indicates 10 μm. (B) Chang and ChangX-34 cells were incubated with MitoTrackerRed for 30 min, and nuclei were subsequently stained with DAPI. The MTOC in ChangX-34 cells was identified by anti-pericentrin immunofluorescence (right panel). (C) Microtubules were depolymerized by the addition of 3.3 μM nocodazole (Noc) for 2 h and repolymerized by the removal of nocodazole by several washes with PBS. Mitochondria and α-tubulin (green) are representative of four independent experiments. (D) Actins were depolymerized by treatment with 1 μM cytochalasin D (Cyt. D) for 1 h and repolymerized by the removal of cytochalasin D. Mitochondria and β-actin (green) staining are representative of three different experiments. The crescent pattern of mitochondrion distribution was used as the criterion for judging mitochondrial clustering.
FIG. 3.
Microtubule-dependent mitochondrial clustering occurs in HBV-replicating Huh7 cells. (A) Subcellular localizations of HBx and mitochondria in Huh7 and HBV-related SNU368 hepatoma cells were visualized by immunofluorescence staining using anti-HBx antibody (green) and MitoTrackerRed (red) under a fluorescence microscope. The scale bar indicates 10 μm. (B) HBV DNA was extracted from isolated core particles and analyzed by Southern blot analysis. Single- and double-stranded linear and partially double-stranded relaxed circular forms of HBV DNA are marked SS, DL, and RC, respectively. An HBV plasmid construct (1 ng) was used as a control marker. (C) Mitochondrial staining in Huh7 and HBV replicating Huh7-HBV cells. (D) Huh7 cells were transfected with a pHBV replicon (pHBV) or pHBV harboring a stop codon right after the AUG of the HBx gene (pHBVX−) using the PEI method, and the subcellular localization of HBx (green) and mitochondria (red) was visualized by immunofluorescence staining. The scale bar indicates 10 μm. (E) Microtubules were depolymerized by the addition of 3.3 μM nocodazole (Noc) for 2 h and repolymerized by the removal of nocodazole by several washes with PBS. Mitochondria and α-tubulin were visualized by MitoTrackerRed and immunofluorescence staining. A representative image of three independent experiments is shown.
FIG. 3.
Microtubule-dependent mitochondrial clustering occurs in HBV-replicating Huh7 cells. (A) Subcellular localizations of HBx and mitochondria in Huh7 and HBV-related SNU368 hepatoma cells were visualized by immunofluorescence staining using anti-HBx antibody (green) and MitoTrackerRed (red) under a fluorescence microscope. The scale bar indicates 10 μm. (B) HBV DNA was extracted from isolated core particles and analyzed by Southern blot analysis. Single- and double-stranded linear and partially double-stranded relaxed circular forms of HBV DNA are marked SS, DL, and RC, respectively. An HBV plasmid construct (1 ng) was used as a control marker. (C) Mitochondrial staining in Huh7 and HBV replicating Huh7-HBV cells. (D) Huh7 cells were transfected with a pHBV replicon (pHBV) or pHBV harboring a stop codon right after the AUG of the HBx gene (pHBVX−) using the PEI method, and the subcellular localization of HBx (green) and mitochondria (red) was visualized by immunofluorescence staining. The scale bar indicates 10 μm. (E) Microtubules were depolymerized by the addition of 3.3 μM nocodazole (Noc) for 2 h and repolymerized by the removal of nocodazole by several washes with PBS. Mitochondria and α-tubulin were visualized by MitoTrackerRed and immunofluorescence staining. A representative image of three independent experiments is shown.
FIG. 4.
Perinuclear clustering of mitochondria occurs in a dynein-dependent manner. (A) Cells were stained with MitoTrackerRed and permeabilized with 0.075% Triton X-100 in PHEM before fixation, and the distribution of the DIC was determined. The fluorescence image was acquired after _Z_-stack scanning. The arrows indicate line scanning (10 μm) starting from the side of the plasma membrane. The lower panel shows the corresponding scanning data. Representative data from three independent experiments are shown. (B) Chang cells were cotransfected with pCDNA3 (control), pMyc-HBx, and/or p50 (dynamitin) expression vectors along with plasmid pEGFP for the identification of transfected cells. The area occupied by mitochondria was quantified according to criteria described in Materials and Methods. Data shown on the right are the means ± standard deviations from two to four separate experiments (**P < 0.001 by Student's t test). (C) A neutralizing antibody (Ab) against DIC or control mouse IgG mixed with green fluorescent protein (GFP) was microinjected into cells (n = 300) using an automatic micromanipulator. Four hours later, cells were treated with 3.3 μM nocodazole for 2 h, washed, and examined for the subcellular localization of the mitochondria using confocal microscopy. White arrowheads indicate cells microinjected with the anti-DIC antibody, and yellow arrows represent neighboring noninjected ChangX-34 cells with clustered mitochondria. Two separate experiments were carried out. (D) ChangX-34 cells were pretreated with 0.5 mM EHNA for 0.5 h, which was followed by the addition of 3.3 μM nocodazole (Noc) for 2 h. Microtubules were repolymerized by the removal of nocodazole, and 0.5 mM EHNA was resupplied for 0.5 h. Mitochondria and α-tubulin (green) were visualized.
FIG. 5.
HBx activates p38 MAPK. (A) Huh7 cells were transfected with the expression vector of HBx (pMyc-HBx) using PEI, and whole-cell lysates were obtained at 24 and 48 h after transfection. Expression levels of the phosphorylated form of p38 (p-p38), total p38, and HBx were determined by Western blotting and quantified using densitometry. The β-actin level indicates the normalization of protein levels in each lane. (B) Huh7 cells were transfected with the expression vector of pCDNA3 or Raf-CA or MKK6 expression vectors, and inductions of the phosphorylated ERK and p38 were determined by Western blotting at 24 h after transfection.
FIG. 6.
Activation of p38 MAPK mediates mitochondrial clustering induced by HBx. (A) Huh7 cells were transfected with the expression vectors of HBx, Raf-CA, or MKK6 along with pEGFP, and the images of MitoTrackerRed-stained cell were obtained after 48 h. The area occupied by mitochondria in a cell was determined according to the criteria described in Materials and Methods. Data shown on the right are the means ± standard deviations from three independent experiments (
P < 0.001 by Student's _t_ test). (B) The specific inhibitor SB203580 (0.5 μM), wortmannin (0.2 μM), or calphostin C (0.25 μM) was applied to ChangX-34 cells right after nocodazole release, and mitochondrial reclustering was examined at 30 and 60 min by MitoTrackerRed staining (data not shown). When the percentage of mitochondrial area per cell was <60%, it was considered to be aggregated cells, and those with >75% were considered to be nonaggregated cells. The bar represents means ± standard deviations from three separate experiments (P < 0.01 and P < 0.001 by Student's t test). Con, control.
References
- 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
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Medical