key: cord-0897583-afulhpin
authors: Lee, H‐R.; Cho, Y. Y.; Lee, G. Y.; You, D‐g.; Yoo, Y. D.; Kim, Y. J.
title: A direct role for hepatitis B virus X protein in inducing mitochondrial membrane permeabilization
date: 2018-01-24
journal: J Viral Hepat
DOI: 10.1111/jvh.12831
sha: 2c6f515782f6691eb9cfef3e60fefd0f0878ea8b
doc_id: 897583
cord_uid: afulhpin

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.

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.

hepatitis B virus X protein, membrane permeabilization, viroporin

Hepatitis B virus (HBV) is a major cause of hepatitis, and nearly 240 million people are infected worldwide. 1 HBV is associated with hepatic fibrosis, cirrhosis and hepatocellular carcinoma. 2,3 HBV is a DNA virus belonging to the Hepadnaviridae family and encodes 7 proteins: 5 structural and 2 nonstructural proteins. The structural proteins include 3 envelope proteins (small, middle and large surface proteins), capsid protein and polymerase. The nonstructural proteins include HBeAg (secreted e antigen) and HBx (hepatitis B virus X protein). 4 HBx is a nonstructural protein with a molecular weight of 17.5 kDa. HBx functions as a multifunctional protein that interacts with various transcription factors and cell cycle-related regulatory proteins, adjusting the intracellular environment to optimal conditions for viral production. [5] [6] [7] Interestingly, its intracellular distribution varies depending on its expression level in cells. For example, HBx was detected in the nucleus at a low expression level, while it was distributed throughout the cytoplasm at a high expression level. 6 HBx has been reported to target the mitochondrial outer membrane, but not the endoplasmic reticulum, the plasma membrane or lysosomes. 6, 8 Mitochondrial outer membranelocalized HBx is known to alter mitochondrial morphology, fission and aggregation. 9 HBx also induces intracellular ROS production 9,10 and mitochondrial depolarization in hepatoma cells, leading to disruption of mitochondrial respiration and apoptosis. HBx induces mitochondrial membrane permeability, resulting in cytochrome c release and mitochondrial Ca 2+ overload that cause apoptosis. 11, 12 Although the role of HBx in the mitochondria is not fully understood, it is clear that mitochondrial outer membrane-localized HBx exerts a different role than nuclear-targeted HBx, which functions in transcriptional activation.

The viruses that cause liver inflammation have been reported to encode viroporins. Hepatitis A virus (HAV) 2B and hepatitis C virus (HCV) p7 directly induce membrane permeabilization, collectively termed viroporins. 13, 14 These are small, nonstructural proteins forming nonselective channels to transport cations and large molecules and have been proposed to modulate the intracellular electrochemical balance of host cells by inducing membrane permeabilization. 13, 15 In a related study, HBV was also shown to induce mitochondrial membrane permeabilization. 16 However, the viral protein responsible for membrane permeabilization in HBV was not yet fully understood.

Here, we explored a novel function for HBx, demonstrating that HBx induces membrane permeabilization independently of mitochondrial proteins.

Egg Lα-phosphatidylcholine (PC), egg Lα-phosphatidylethanolamine (PE), liver Lα-phosphatidylinositol (PI), brain Lα-phosphatidylserine 

The HBx protein was obtained as described previously with some modifications. 17 Briefly, pET28A-6x his tag-HBx was transformed into Escherichia. coli BL21 (DE3) and incubated with 0.5 mmol L −1 isopropyl-1-thioβ-D-galactoside (IPTG) for 4 hours at 25°C. Inclusion bodies were harvested and lysed with 8 mol L −1 urea, 10 mmol L −1 dithiothreitol (DTT) and 100 mmol L −1 Tris/NaOH (pH 8) overnight.

The HBx protein was purified by nickel-nitrilotriacetate (Ni-NTA) agarose (Qiagen, Valencia, CA, USA) and then purified by a PD-10 desalting column (GE Healthcare, Buckinghamshire, UK). Purified 6x his tag-HBx was refolded as described previously. 17 

HBx has mainly been studied as a transactivation factor, which interacts with several cellular proteins in host cells. However, the function of HBx on the mitochondrial outer membrane has not been well evaluated. As the exact function of HBx on the mitochondrial membrane is challenging to evaluate in cells, we attempted to investigate HBx function using a recombinant HBx protein and an artificial membrane system. We synthesized recombinant HBx protein in E. coli and recovered its biological activity through a rational refolding method, as previously reported. 17 The purity of the refolded recombinant HBx protein was examined via Coomassie blue staining (>95%), and it was further confirmed by anti-HBx and anti-His antibodies ( Figure 1A ,B). As HBx was shown to be inserted into the mitochondrial outer membrane via its transmembrane domain (residues 53-70), we generated liposomes with a rat liver mitochondrial outer membrane composition, as previously reported. 18, 19 To observe the HBx localization in liposomes, we prepared mitochondrial outer membrane-mimic giant unilamellar vesicles (MOM-GUVs) including ATTO 390-DOPE. We treated the recombinant HBx protein for 2 minutes, and HBx-targeted MOM-GUVs were then stained with nickel-nitrilotriacetate conjugated to ATTO 488 fluorescent dye (NTA-ATTO 488) for the detection of poly-Histagged HBx by fluorescence microscopy. As expected, the recombinant HBx protein was shown to target the MOM-GUVs ( Figure 1C ).

HBx triggers membrane depolarization, probably through interaction with the mitochondrial permeability transition pore. 20 In this study, we investigated HBx-induced membrane permeabilization using MOM-GUVs without any mitochondrial proteins. First, we created MOM-GUVs with 200 mmol L −1 sorbitol solution, and they were added to the external buffer containing 100 mmol L −1 KCl and 10 mmol L −1 HEPES/KOH (pH 7.4). Using phase contrast microscopy, we found that MOM-GUVs became transparent after treatment with HBx in a time-dependent or concentration-dependent manner (Figure 2A To quantify the HBx-induced membrane permeabilization, we performed a liposome permeabilization assay; these are generally used in viroporin studies to evaluate pore-forming activity. 21 As CF is self-quenched at high concentrations and becomes fluorescent at low concentrations, we made CF-LUVs with the lipid composition of the mitochondrial outer membrane. Recombinant HBx protein added to CF-LUVs caused liposome permeabilization in a time-dependent or concentration-dependent manner, similar to its activity on GUVs ( Figure 3A ,B). We also tested the HCV p7 protein (a known cause of membrane permeabilization) as a positive control. 19, 22 These results indicated that HBx directly induces membrane permeabilization, independently of other cellular proteins.

We hypothesized that the liposome permeabilization induced by HBx could be nonselective permeabilization by protein-lipid pore formation, similar to the activity of some viroporins (eg the severe acute respiratory syndrome coronavirus E protein). 23 In most cases, viroporin-induced pore formation is nonselective or weakly selective for cations. 23 Therefore, we examined the inhibitory effect of gadolinium ion (Gd 3+ ) and lanthanum ion (La 3+ ), both known as nonselective channel blockers. 24, 25 Interestingly, Gd 3+ and La 3+ effectively inhibited HBx-induced liposome permeabilization, but Ca 2+ and Mg 2+ ions did not ( Figure 4A,B) . The inhibitory effect of Gd 3+ ( Figure 4C) and La 3+ ( Figure 4D ) was observed in a concentration-dependent manner.

We next examined whether the liposome permeabilization caused 

In this study, we showed that NN-DNJ inhibited HBx-induced liposome permeabilization. Next, we attempted to examine the NN-DNJ effect on HBx-induced mitochondrial depolarization in Huh-7 cells.

We transfected HBx into Huh-7 cells for 6 hours and treated cells with NN-DNJ for an additional 18 hours ( Figure 5A ). Mitochondrial membrane potential was quantified using TMRE, a mitochondrial membrane potential indicator. We found that HBx-induced mitochondrial depolarization was inhibited by NN-DNJ ( Figure 5B,C) , and NN-DNJ did not change the expression level of HBx ( Figure 5D ). As demonstrated in Figure 4E , amantadine and rimantadine showed no significant inhibitory effects ( Figure 5E ). These results indicate that HBx-induced mitochondrial depolarization could be inhibited by NN-DNJ, and this may be related to the membrane permeabilization induced by HBx.

We investigated the specific region of HBx that accounted for liposome permeabilization. We hypothesized that HBx-induced membrane permeabilization was not dependent on the transactivation region. Considering that the hydrophobic region of cytochrome c has been shown to insert into membranes to form lipidic pores, we syn- 

Viruses alter the function of cellular organelles to enhance viral replication and escape from host cells. One of these organelles is the mitochondrion, and the modulation of this organelle is indispensable for the viral life cycle. 26 Mitochondria have a diverse set of functions including energy production, apoptosis and metabolite supply. 27 One of the main events during apoptosis is mitochondrial depolarization, which is caused by the opening of a permeability transition (PT) pore. 20 The PT pore includes adenine nucleotide translocator (ANT) protein, voltagedependent anion channel (VDAC) protein and matrix cyclophilin D protein. 28 A variety of viruses control the PT pore to trigger apoptosis.

to decrease mitochondrial membrane permeability (MMP), inducing apoptosis by the release of pro-apoptotic proteins. 29 The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr), the Walleye dermal sarcoma virus Orf C protein and the human T-lymphotropic virus type 1 (HTLV-1) p13II protein have all been reported to localize in the mitochondria and modulate mitochondrial proteins to induce apoptosis. [30] [31] [32] In contrast, some viruses directly induce mitochondrial depolarization through viral-encoded viroporins. HCV p7, human Tcell leukaemia virus type 1 (HTLV-1) p13 and picornavirus 2B proteins detected in mitochondria all induce mitochondrial depolarization. 33, 34 In the case of HCV p7, the protein forms hexameric complexes that act as ion channels permeable to cations to enhance the entry and escape of viral particles. 19, 35, 36 HBx also reduces the transmembrane potential and induces apoptosis by interacting with VDAC3. 20 HBx mainly has been studied in terms of its regulation of signalling cascades, including its interaction with several cellular proteins.

In this study, we evaluated the function of HBx on biological membranes and demonstrated that HBx induces membrane permeabilization. This effect was inhibited by a viroporin inhibitor and by nonselective cation channel blockers ( Figure 4) . As an artificial membrane system without any cellular proteins was used, we hypothesize that this membrane permeabilization is mediated by HBx only. We excluded the possibility of HBx as an ion channel protein (viroporin) because its predicted secondary structure is far different from that of the viroporins, which have 1 or 2 alpha-helical transmembrane domains. We questioned the means by which HBx could trigger membrane permeabilization in this artificial liposome system, but to elucidate the exact mechanism, more extensive studies are required. However, we suggest 1 possibility based on the previous studies in which cytochrome c, the protein of the mitochondrial electron transport chain, was reported to induce membrane permeabilization and it shows (or was suggested to have) the "channel-pore dualism," which is regarded as a viroporin phenomenon. 37, 38 We found that HBx permeated CF, which has a 0.6-nm Stokes radius (Figure 3 ). However, HBx is not classified as an ion channel protein. 38 Cytochrome c interacts with specific lipids of the mitochondrial inner membrane to induce lipid pore formation. 38 It was thus shown to induce membrane permeabilization, releasing CF and 10-kDa dextran from liposomes. It has been proposed that the hydrophobic region of cytochrome c affects the formation of lipid pores by its insertion into the acyl chains of the lipid bilayer.

It is possible that HBx forms a protein-lipid pore with viroporinlike activity, and that it induces membrane permeabilization similar to cytochrome c. Consistent with the structural features of cytochrome c, HBx also has a hydrophobic region (residues 54-70) that may serve as a transmembrane domain for insertion into the mitochondrial outer membrane. It is possible that membrane permeabilization by HBx is similar to that of cytochrome c. These results indicate that HBx may form a nonselective pore to pass ions and small molecules through the mitochondrial outer membrane.

However, the mitochondrial outer membrane has a permeability threshold that allows molecules below 1.5 kDa to pass via poreforming channels (such as those created by VDAC). 39 

Rim. Ama.

Rim.

In this study, we tested 3 viroporin inhibitors (amantadine, rimantadine and NN-DNJ). Interestingly, HBx-induced membrane permeabilization was not inhibited by adamantane derivatives (amantadine and rimantadine), whereas NN-DNJ inhibited HBx-induced membrane permeabilization independently of the mitochondrial permeability transition pore complex ( Figure 4 ). Membrane permeabilization induced by the HBx protein was observed in the artificial membrane system, and it now needs to be confirmed in cells. However, it is difficult to confirm this finding in cells because the mechanism behind HBx-induced mitochondrial dysfunction is very complex and has not been well elucidated yet. Instead, we showed that NN-DNJ inhibited HBx-caused mitochondrial depolarization in Huh-7 cells ( Figure 5 ).

Extensive studies remain to fully explain the direct role of HBx in triggering mitochondrial depolarization.

NN-DNJ has low cytotoxicity and has potential to be a therapeutic agent for HBV. 40, 41 NN-DNJ has been used to inhibit HBV, and its inhibitory concentration 50 (IC 50 ) is in the range of 1-10 μM.

Similarly, our results showed that 10 μM of NN-DNJ was sufficient to inhibit HBx-induced mitochondrial depolarization. From the combined results, we can suggest that the NN-DNJ effect on HBV may be related to membrane permeabilization by HBx, and additional experiments to support this hypothesis are needed. In conclusion, we have demonstrated that HBx induces membrane permeabilization, and that this effect is inhibited by 1 viroporin inhibitor and by 2 triply-charged lanthanides. It is possible that other viroporin inhibitors or nonselective channel blockers also inhibit HBx activity. In the present study, we identified a novel function for HBx, demonstrating its direct role in inducing mitochondrial membrane permeabilization. Based on the results obtained in this study, we propose that HBx-induced membrane permeabilization may be an attractive therapeutic target for HBV treatment. 

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The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors

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Hepatitis C virus induces the mitochondrial translocation of Parkin and subsequent mitophagy

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The C-terminal region of the non-structural protein 2B from Hepatitis A Virus demonstrates lipid-specific viroporin-like activity

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Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential

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