Figure 1.
HBV/HBx promotes mitochondrial fission via Drp1 stimulation and activation.
(A and B) Confocal microscopy images showing mitochondrial fragmentation in HBV- and HBx-expressing cells, respectively. Huh7 cells were transiently transfected with HBV (A) or the HBx-flag construct (B). At 2 days post-transfection, cells prestained with MitoTracker (Mito, white) were immunostained with anti-HBsAg (A, green) and anti-flag (B, green) antibodies, respectively. In the zoomed images, typical tubular mitochondria in untransfected cells and fragmented mitochondria in transfected cells are shown. (C) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct were analyzed by Western blotting with antibodies against the indicated proteins. β-actin was used as an internal loading control. Mitochondrial fraction (bottom panel) isolated from HepAD38 cells (see the purity in Figure 2B) was analyzed by Western blotting with phospho-Drp1 (Ser616) antibody. (D–F) Confocal immunofluorescence showing mitochondrial translocation of Drp1 in HBV- and HBx-expressing cells, respectively. Huh7 cells transfected with HBV (D and E) or HBx-flag construct (F) were immunostained with antibodies specific to TOM20 (red), Drp1 (D, green), and phospho-Drp1 (Ser616) (E and F, green), respectively. HBV and HBx gene expression (white) was verified using anti-HBsAg (D and E) and anti-flag (F) antibodies, respectively. In the zoomed images, the yellow color indicates the merge of Drp1 or phospho-Drp1 (Ser616) with mitochondria. (A, B, D, E, and F) Transfected (+) and untransfected (−) cells are marked. Nuclei were stained with DAPI (blue).
Figure 2.
HBV induces mitochondrial translocation of Parkin and stimulates mitophagy-related genes.
(A) Huh7 cells transfected with HBV construct were prestained with MitoTracker (Mito, red) and subsequently, immunostained with anti-Parkin (green) and anti-HBsAg (white) antibodies. Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, yellow color indicates the accumulation of endogenous Parkin recruited to the mitochondria. Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in HBV-expressing cells. (B) The cytosolic (Cyto) and mitochondrial (Mito) fractions were isolated from HepAD38 cells grown in the absence or presence of tetracycline for 48 h. Cellular fractions were analyzed by Western blotting with antibodies specific for the indicated proteins. Fractions: WCL, whole cell lysates; Cyto, purified cytoplasm; Mito, purified mitochondria. Organelle markers: VDAC1, mitochondria; GAPDH, cytoplasm. (C) Parkin protein in whole cell lysates of HBV-expressing cells was immunoprecipitated by anti-Parkin antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal rabbit IgG was used as a control for immunoprecipitation (IP). Western blot analysis for β-actin indicates equivalent amount of cell lysates for IP (C and D). (D) Mfn2 protein in whole cell lysates extracted from HepAD38 cells transfected with non-targeting (NT) and Parkin (P) siRNA, respectively, for 48 h was immunoprecipitated by anti-Mfn2 antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal mouse IgG was used as a control for immunoprecipitation (IP). The protein expression was analyzed by Western blotting with antibodies specific to Mfn2, Parkin, and β-actin proteins. (E and F) HepAD38 and HepG2 cells were grown in the absence or presence of tetracycline (1 µg/ml) for 12 h. (E) Intracellular mRNA levels of Parkin, PINK1, and LC3B were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in Parkin, PINK1, and LC3B gene expression. (F) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins. β-actin was used as a loading control (F and G). The relative intensity of ATF4, Parkin, PINK1, and LC3B protein expression normalized to β-actin was analyzed by ImageJ software. (G) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct for 48 h were analyzed by Western blotting with antibodies specific to the indicated proteins.
Figure 3.
(A) Huh7 cells transiently expressing GFP-LC3 protein were transfected with HBV construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells were immunostained with antibodies against HBsAg (white), TOM20 (blue), and Parkin (red). Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin. (B and C) Quantification of the number of total GFP-LC3 puncta (B) and GFP-LC3 puncta colocalized with TOM20 (C) in the panel (A). (D) Quantitative analysis of the area of GFP-LC3 puncta (white) representing merge of GFP-LC3 puncta, TOM20, and Parkin in the panel (A).
Figure 4.
HBV/HBx induces mitophagolysosomes.
(A) Novel system for monitoring mitophagic flux using dual fluorescence p-mito-mRFP-EGFP reporter (pAT016). Lysosomal delivery of the tandem fusion protein Mito-mRFP-EGFP along with entire mitochondria results in differential quenching and degradation of the two individual fluorochromes, thus allowing for visual analysis of mitophagic flux. (B) Huh7 cells transiently expressing Mito-mRFP-EGFP were transfected with HBV, HBx-flag, and HBV-ΔX constructs, respectively, for 48 h. Cells were immunostained with antibodies specific to HBsAg and flag (white), respectively. Nuclei were stained with DAPI (blue). Transfected (+) and untransfected (−) cells are marked. In the zoomed images, fluorescence signals indicate the expression of Mito-mRFP-EGFP targeting mitochondria: yellow color, no mitophagy; red color, mitophagy. (C) Quantitative analysis of the fluorescence signal targeted to mitochondria in the panel (B). (D) GFP-LC3-expressing Huh7 cells were transfected with HBV construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells prestained with LysoTracker (Lyso, red) were immunostained with antibodies against HBsAg (white) and TOM20 (blue). Nuclei are demarcated with white dot circles. Transfected (+) and untransfected (−) cells are marked. In the zoom images, the arrows denoting white puncta indicate GFP-LC3 puncta (green) colocalized with TOM20 and lysosome. (E) Quantification of the colocalization of GFP-LC3 puncta containing lysosome with mitochondria in the panel (D).
Figure 5.
Attenuation of mitochondrial apoptosis by HBV.
(A–C) Parkin silencing accelerates HBV-induced mitochondrial apoptotic signaling. HepG2 and HepAD38 cells grown in the absence or presence of tetracycline were transfected with non-targeting (NT) and Parkin siRNA, respectively, for 72 h. Cells were used for Western blot analysis using antibodies specific to the indicated proteins (A), caspase-3/7 activity assay (B), and TUNEL assay (C). (D) A model elucidating HBV-induced aberrant mitochondrial dynamics in mitochondrial apoptosis. HBV induces immoderate stimulation in mitochondrial fission and mitophagy through mitochondrial translocation of Drp1 and Parkin, respectively, eventually causing disruption in mitochondrial dynamics. Inhibition of mitophagy by Parkin silencing can lead to apoptotic cell death in HBV-infected host cells via accumulation of apoptotic signal (red).