A direct role for hepatitis B virus X protein in inducing mitochondrial membrane permeabilization

Abstract

Hepatitis B virus X protein (HBx) acts as a multifunctional protein that regulates intracellular signalling pathways during HBV infection. It has mainly been studied in terms of its interaction with cellular proteins. Here, we show that HBx induces membrane permeabilization independently of the mitochondrial permeability transition pore complex. We generated mitochondrial outer membrane-mimic liposomes to observe the direct effects of HBx on membranes. We found that HBx induced membrane permeabilization, and the region comprising the transmembrane domain and the mitochondrial-targeting sequence was sufficient for this process. Membrane permeabilization was inhibited by nonselective channel blockers or by N-(n-nonyl)deoxynojirimycin (NN-DNJ), a viroporin inhibitor. Moreover, NN-DNJ inhibited HBx-induced mitochondrial depolarization in Huh-7 cells. Based on the results of this study, we can postulate that the HBx protein itself is sufficient to induce mitochondrial membrane permeabilization. Our finding provides important information for a strategy of HBx targeting during HBV treatment.

Keywords: hepatitis B virus X protein; membrane permeabilization; viroporin.

Figure 1

Targeting of the recombinant HBx protein to MOM‐GUVs. A‐B, The purified recombinant 6x‐His‐HBx protein was analysed by Coomassie blue staining and by immunoblot with an anti‐HBx antibody or anti‐His antibody. C, GUVs were prepared at a 54:29:13:2:1:1 molar ratio of PC:PE:PI:PS:PA:CL with 0.5% ATTO 390‐DOPE for the fluorescent labelling of membranes. HBx was treated to GUVs for 5 min, and HBx‐targeted GUVs were incubated with NTA‐ATTO 488 for 5 min to visualize 6x‐His‐HBx on the membranes. The analysis was performed by fluorescence microscopy. The scale bar represents 10 μm

Figure 2

GUV permeabilization induced by HBx. MOM‐GUVs were mixed with external buffer [100 mmol L⁻¹ KCl and 10 mmol L⁻¹ HEPES/KOH (pH 7.4)] and treated with the indicated concentrations of recombinant HBx. A, Images were captured by phase contrast microscopy in a time‐dependent manner. The scale bars represent 50 μm. B, The indicated square regions were magnified. The scale bars represent 10 μm. C, Line profiles of HBx‐treated MOM‐GUVs. The lines represent cross sections of GUVs, and data were analysed by ImageJ software

Figure 3

Quantification of HBx‐induced liposome permeabilization. A, Recombinant HBx was added to CF‐enclosed MOM‐LUVs, and the mixture was analysed by spectrophotometry over a time course to measure CF release. B, HBx‐induced liposome permeabilization was quantified at 20 min. HCV p7 was used for a positive control

Figure 4

Inhibitory effects of nonselective channel blockers and viroporin inhibitors on HBx‐induced liposome permeabilization. A, CF‐enclosed MOM‐LUVs were incubated with the indicated ions (100 nmol L⁻¹) after exposure to recombinant HBx (20 nmol L⁻¹). B, HBx‐induced liposome permeabilization was quantified at 20 min. Concentration‐dependent inhibitory effects of GdCl₃ (C) or LaCl₃ (D) on HBx‐induced liposome permeabilization were quantified at 20 min. E, CF‐enclosed MOM‐LUVs were incubated with the indicated viroporin inhibitors (1 μM) after exposure to recombinant HBx (20 nmol L⁻¹) for 20 min. F, Concentration‐dependent inhibitory effects of NN‐DNJ on HBx‐induced liposome permeabilization were quantified at 20 min. **P ≤ 0.01, ***P ≤ 0.001 vs. control by two‑way analysis of variance (ANOVA). N.S.; not significant, Aman., Amantadine; Riman., Rimantadine

Figure 5

Inhibitory effect of NN‐DNJ on HBx‐induced mitochondrial depolarization. A, Flag HBx was transfected for 6 hrs, and NN‐DNJ was added for an additional 18 hrs. B, C, E, After transfection with vector or HBx for 6 hrs, the Huh‐7 cells were treated with viroporin inhibitors for 18 hrs and then stained with TMRE. Decreased levels of ∆Ψm were analysed by flow cytometry. D, Cell lysates were analysed by immunoblot with anti‐Flag antibody. *P ≤ 0.05, **P ≤ 0.01 vs. control by two‑way analysis of variance (ANOVA). N.S.; not significant, Cont., Control; Ama., Amantadine; Rim., Rimantadine

Figure 6

Effect of HBx residues 53‐117 on liposome permeabilization. A‐B, A deletion mutant of HBx (53‐117) containing the transmembrane domain and mitochondrial‐targeting sequence was added to CF‐enclosed MOM‐LUVs and analysed by spectrophotometry over a time course to measure CF release. CF‐enclosed MOM‐LUVs were incubated with (C) the indicated nonselective channel blockers (100 nmol L⁻¹) or (D) NN‐DNJ after exposure to the 53‐117 mutant (20 nmol L⁻¹) for 20 min, and CF release was measured by spectrophotometry *P ≤ 0.05, ***P ≤ 0.001 vs. control by two‑way analysis of variance (ANOVA).