Inhibition of HBV Transcription From cccDNA With Nitazoxanide by Targeting the HBx-DDB1 Interaction - PubMed (original) (raw)

Inhibition of HBV Transcription From cccDNA With Nitazoxanide by Targeting the HBx-DDB1 Interaction

Kazuma Sekiba et al. Cell Mol Gastroenterol Hepatol. 2019.

Abstract

Background & aims: Hepatitis B virus (HBV) infection is a major health concern worldwide. Although currently used nucleos(t)ide analogs efficiently inhibit viral replication, viral proteins transcribed from the episomal viral covalently closed circular DNA (cccDNA) minichromosome continue to be expressed long-term. Because high viral RNA or antigen loads may play a biological role during this chronicity, the elimination of viral products is an ultimate goal of HBV treatment. HBV regulatory protein X (HBx) was recently found to promote transcription of cccDNA with degradation of Smc5/6 through the interaction of HBx with the host protein DDB1. Here, this protein-protein interaction was considered as a new molecular target of HBV treatment.

Methods: To identify candidate compounds that target the HBx-DDB1 interaction, a newly constructed split luciferase assay system was applied to comprehensive compound screening. The effects of the identified compounds on HBV transcription and cccDNA maintenance were determined using HBV minicircle DNA, which mimics HBV cccDNA, and the natural HBV infection model of human primary hepatocytes.

Results: We show that nitazoxanide (NTZ), a thiazolide anti-infective agent that has been approved by the FDA for protozoan enteritis, efficiently inhibits the HBx-DDB1 protein interaction. NTZ significantly restores Smc5 protein levels and suppresses viral transcription and viral protein production in the HBV minicircle system and in human primary hepatocytes naturally infected with HBV.

Conclusions: These results indicate that NTZ, which targets an HBV-related viral-host protein interaction, may be a promising new therapeutic agent and a step toward a functional HBV cure.

Keywords: Drug Screening; Minicircle; Primary Hepatocyte Infection.

Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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Graphical abstract

Figure 1

Figure 1

High-throughput screening system to monitor HBx–DDB1 binding. (A) The principle underlying detection of the HBx–DDB1 interaction using split luciferase. The separated NanoBiT subunits, LgBit and SmBit, associate weakly. However, once the target proteins to which the NanoBiT subunits are tagged interact, the luminescent complex is assembled and easily detected with the luciferase assay. (B) All possible combinations of the established constructs, which were used to determine the optimal tag positions and usage of LgBit or SmBit. (C) HBx fused to LgBit at its C terminus (HBx-LgBit) and DDB1 fused to SmBit at its N terminus (SmBit-DDB1) made up the best pair, which produced the brightest relative luciferase signal after transfection. Halo-tag-SmBit was included as a negative control. Data represent the mean (n = 3) ± SD of triplicate experiments. ***P < .0001 (t test). (D) Z’ scores at each time point during the course of the assays. (E, F) Time course of luciferase activity in (C) HEK293T and (D) HepG2 cells after addition of 5 candidate compounds selected from the initial screening. Data represent the mean (n = 3) ± SD of triplicate experiments. LOP, loperamide; PMZ, pimozide; TRF, toremifene; VBL, vinblastine. (G, H) Dose dependency of the inhibitory effect of NTZ on the HBx–DDB1 interaction in (E) HEK293T and (F) HepG2 cells. Data show the percentage of control results for luciferase activity 30 minutes after addition of NTZ. Experiments were performed in triplicate (n = 3). (I) Cell toxicity was determined with different doses of NTZ. HepG2 or HepG2 cells that constitutively express Flag-HBx protein (HepG2Flag-HBx) cells were treated with the indicated doses of NTZ for 24 hours. Cell viability was determined through a cell counting assay. Data show the results of triplicate experiments (n = 3).

Figure 2

Figure 2

NTZ inhibits the HBx–DDB1 interaction. (A) HEK293T cells were transfected with plasmid expressing Flag-HBx, and treated with NTZ (10 μM) or DMSO (as a control) for 24 hours. Inhibition of the HBx–DDB1 interaction was examined through IP using anti-FLAG antibody, followed by Western blot analysis. Five percent of the total cell lysate was used as “input.” Representative results from 3 independent experiments are shown. Summarized results (n = 3) of relative band intensity are shown below the panels. *P = 6.8 × 10–5 (t test). (B) Inhibitory effects of the 5 candidate compounds on the HBx–DDB1 interaction. HEK293T cells were transfected with a plasmid expressing Flag-HBx, and treated with the indicated compounds (10 μM) for 24 hours. Inhibition of the HBx–DDB1 interaction was examined by IP using anti-FLAG antibody, followed by Western blot analysis. Representative results of 3 independent experiments are shown. Summarized results of relative band intensity are shown below the panels (n = 3). *P = 1.2× 10–3; **P = 2.1 × 10–3; ***P = 1.5 × 10–4 (t test). (C) HepG2 cells were transfected with a plasmid expressing Flag-HBx, and treated with NTZ (10 or 20 μM) or DMSO (as a control) for 24 hours. IP and Western blotting were performed as described in panel A. Representative results from 3 independent experiments are shown. Summarized results of relative band intensities are shown below the panels (n = 3). NS, not significant; *P = 1.3 × 10–3; **P = 1.4 × 10–4 (t test).

Figure 3

Figure 3

Inhibitory effects of NTZ on the HBx–DDB1 interaction in vitro. GST-tagged recombinant DDB1 protein and untagged recombinant HBx protein were mixed in vitro. NTZ or DMSO was added to the mixture and incubated for 20 minutes, followed by pull-down using anti-GST antibody and Western blotting to determine the levels of DDB1 and HBx in the pulled-down samples. Representative results of 3 independent experiments are shown. Summarized results of relative band intensities are shown below the panels (n = 3). NS, not significant; *P = 7.1 × 10–5 (t test).

Figure 4

Figure 4

NTZ reverses the degradation of Smc5 induced by HBx. (A) HepG2 and (B) HepG2FlagHBx cells, which stably express Flag-HBx protein, were treated with NTZ or DMSO for 48 hours. The expression level of Smc5 protein was determined by Western blotting. Representative results from 3 independent experiments are shown. Summarized results of relative band intensities (n = 3) are shown below the panels. NS, not significant; *P = 5.4 × 10–3; **P = 1.1 × 10–5 (t test).

Figure 5

Figure 5

NTZ inhibits viral RNA transcription. (A) HepG2 cells were transfected with mcHBV-Gluc and a control plasmid, pCMV-Cluc. After 3 days, NTZ (10 or 20 μM) or DMSO (as a control) was added. Five days after transfection, the culture medium was collected for analysis of Gluc and Cluc activities. Relative Gluc levels were calculated after normalization to Cluc values. Data represent the mean (n = 3) + SD of triplicate experiments. *P = 2.0 × 10–3; **P = 9.1 × 10–4 (t test). (B) HepG2 cells were transfected with mcHBV-Gluc. Four days after transfection, NTZ (10 μM) or control DMSO treatment was initiated. Three days later, Gluc activity in the culture media was assayed (left panel). To confirm equal Gluc minicircle DNA levels, levels in the cells were determined through qPCR and relative levels are shown. Data represent the mean (n = 3) + SD of triplicate experiments. *P = 8.5 × 10–6; NS, not significant (t test). (C) HepG2 cells were transfected with mcHBV-Gluc. Four days after transfection, NTZ (10 μM), IFNα2a (1.0 × 103 or 5.0 × 103 units/mL), or control DMSO treatment was started. Two days later, relative Gluc activities in the culture media were calculated after normalization using intracellular Gluc DNA levels. Data represent the mean (n = 3) + SD of triplicate experiments. *P = 2.8 × 10–3; **P = .016; ***P = 1.0 × 10–3; ****P = 1.2 × 10–3; *****P = 8.2 × 10–4; NS, not significant (t test). (D) HepG2 or HepG2Flag-HBx cells were transfected with mcHBV-Gluc (WT) or mcHBV-Gluc-ΔX (ΔX) as indicated and a control plasmid, pCMV-Cluc. After 3 days, NTZ (10 μM) or DMSO (as a control) was added. Five days after transfection, the culture medium was collected for analysis of Gluc and Cluc activities. Relative Gluc levels were calculated after normalization to Cluc values. Data represent the mean (n = 3) + SD of triplicate experiments. *P = 1.1 × 10–3; NS, not significant (t test). (E, F) HepG2 cells were transfected with mcHBV-Gluc. Six days after transfection, NTZ (10 μM) or DMSO was added. Culture medium was renewed every 2 days. Twelve days after transfection, total RNA was extracted and qPCR was performed to measure (E) total HBV mRNA levels and (F) 3.5-kb mRNA levels. Data represent the mean + SD of triplicate experiments. *P = 2.7 × 10–4, **P = 1.2 × 10–2. (G, H) HepG2FlagHBx cells were transfected with mcHBV-Gluc. Three days after transfection, cells were passaged and treated with NTZ (10 μM) or DMSO for another 2 days. (G) Five days after transfection, Gluc activities in the culture media were assayed. (H) Simultaneously, total RNA was extracted and qPCR was performed to measure 3.5-kb mRNA levels. Data represent the (G) mean (n = 3) ± SD or (H) mean (n = 3) + SD of triplicate experiments. *P = 3.7 × 10–6; **P = 7.0 × 10–4 (t test). (I, J) HepG2 cells were transfected with minicircle HBV (genotype D). Five days after transfection, NTZ (at the indicated doses) or DMSO (as a control) was added. Culture medium was renewed every 2 days. Eleven days after transfection, total RNA was extracted and qPCR was performed to measure (I) total HBV mRNA and (J) 3.5-kb mRNA levels. Data represent the mean (n = 3) + SD of triplicate experiments. *P = 9.7 × 10–3; ∗∗P = 2.2 × 10–3; ∗∗∗P = 1.1 × 10–2; ∗∗∗∗P = 2.4 × 10–4 (t test).

Figure 6

Figure 6

NTZ inhibits HBV mRNA transcription in HepAD38 cells. HepAD38 cells were cultured for 2 days after addition of tetracycline to stop HBV transcription from the genome. NTZ (10 μM) or DMSO was added, and culture media containing tetracycline was renewed every 2 days. After 5 days, qPCR was conducted to measure HBV 3.5-kb mRNA levels. Data represent the mean (n = 3) + SD of triplicate experiments. *P = 7.3 × 10–5 (t test). A positive control (PC) included HepAD38 cells cultured without tetracycline for a long time, allowing for constitutive HBV RNA transcription from the genome.

Figure 7

Figure 7

NTZ decreases viral protein and cccDNA levels. (A) HepG2 cells were transfected with minicircle HBV (genotype D). Four days after transfection, NTZ (at the indicated doses) or control DMSO was added. Culture medium was renewed every 2 days. Eleven days after transfection, cell lysates were subjected to Western blotting to determine the indicated protein levels. Representative results of 3 independent experiments are shown. Summarized data (n = 3) of relative band intensities are shown below the panels. NS, not significant: *P = 1.6 × 10–3; **P = 1.1 × 10–3; ***P = 1.6 × 10–3; ****P = 1.1 × 10–3 (t test). (B) HBV cccDNA levels were determined through ddPCR and Southern blotting. HepG2 cells were transfected with minicircle HBV (genotype D). Three days after transfection, NTZ (10 μM) or control DMSO was added. Five days after transfection, DNA extracted using the Hirt method was subjected to Southern blotting and ddPCR. For Southern blotting, control DNA from HBV transfected- and DMSO-treated cells was subjected to heat denaturing or heat denaturing with EcoRI digestion. Heat denaturing turns relaxed circular DNA and double-stranded linear DNA into single-stranded DNA, but does not affect cccDNA. EcoRI after heat denature cuts cccDNA into double-stranded linear DNA, but does not affect single-stranded DNA, confirming the integrities of the corresponding bands. Images of the cccDNA bands acquired with a longer exposure are shown in the lower panel. For ddPCR, the same samples subjected to Southern blotting were used for quantitation of cccDNA levels. Data represent the mean (n = 3) + SD of triplicate experiments. *P = 8.5 × 10–4 (t test). (C) Correlation between cccDNA levels in Southern blotting and ddPCR results. HepG2 cells were transfected with minicircle HBV (genotype D). Five days after transfection, DNA extracted using the Hirt method was subjected to Southern blotting and ddPCR with the indicated amounts of plasmid-safe digested DNA. The results of Southern blotting and ddPCR confirmed similar trends.

Figure 8

Figure 8

Effects of NTZ in a natural HBV infection model. (A, B) Human primary hepatocytes were infected with HBV (genotype C). At 8 days after infection and before NTZ or DMSO were added, 1 mL of culture medium was collected for measurement of (A) HBsAg levels and (B) HBV-DNA) to confirm that the established infection load was equally distributed. Data represent the mean (n = 3) + SD of triplicate experiments. NS, not significant; NC, negative control without HBV infection. (C) Human primary hepatocytes were treated with the indicated doses of NTZ for 72 hours. Cell viability was determined through a cell counting assay. Data (n = 3) show the results of triplicate experiments. (D) Human primary hepatocytes were infected with HBV (genotype C). Eight days after infection, NTZ (20 μM) or DMSO was added. Culture medium was renewed every 24 hours. After treatment for 5 days, Smc5 protein levels in the cell lysates were determined through Western blotting. Representative results of 3 independent experiments are shown. Summarized results (n = 3) of relative band intensities are shown below the panels. *P = 7.3 × 10–5 (t test). (E, F) HBV infection and NTZ addition were performed as described in panel D. (E) Total viral mRNA and (F) 3.5-kb mRNA levels were determined by qPCR. Data represent the mean (n = 3) + SD of triplicate experiments. *P = 3.3 × 10–3; **P = 3.8 × 10–4 (t test). (G, H) After HBV infection and NTZ treatment as described in panel D, HBsAg levels in 1 mL of culture media were measured using (G) an enzyme-linked immunosorbent assay and (H) preS2 protein levels in cell lysates were determined through Western blotting. Data represent the mean (n = 3) + SD of triplicate experiments. *P = 3.4 × 10–3 (t test). (I) After HBV infection and NTZ treatment as described in panel D, cccDNA was isolated using plasmid-safe DNase treatment of total DNA extracted from the cells. cccDNA levels were determined via ddPCR. n.d., not detected in noninfected cells (NC). Data represent the mean (n = 3) + SD of triplicate experiments. *P = 6.7 × 10–4 (t test).

Figure 9

Figure 9

Effects of NTZ in the early stage of natural HBV infection model. Human primary hepatocytes were infected with HBV (genotype C). Two days after infection, NTZ (20 μM) or DMSO was added. Culture medium was replaced every 24 hours. After treatment for 5 days, culture medium and cell lysates were collected for the following analyses. (A) Total viral mRNA and (B) 3.5-kb mRNA levels were determined by qPCR. Data represent the mean (n = 3) + SD of triplicate experiments. *P = 2.7 × 10–5; **P = 1.7 × 10–8 (t test). (C) HBs antigen levels in 1 mL of culture media were measured using an enzyme-linked immunosorbent assay. Data represent the mean (n = 3) + SD of triplicate experiments. *P = 3.6 × 10–4 (t test).

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