Control of APOBEC3B induction and cccDNA decay by NF-κB and miR-138-5p

Abstract

Background & aims: Immune-mediated induction of cytidine deaminase APOBEC3B (A3B) expression leads to HBV covalently closed circular DNA (cccDNA) decay. Here, we aimed to decipher the signalling pathway(s) and regulatory mechanism(s) involved in A3B induction and related HBV control.

Methods: Differentiated HepaRG cells (dHepaRG) knocked-down for NF-κB signalling components, transfected with siRNA or micro RNAs (miRNA), and primary human hepatocytes ± HBV or HBVΔX or HBV-RFP, were treated with lymphotoxin beta receptor (LTβR)-agonist (BS1). The biological outcomes were analysed by reverse transcriptase-qPCR, immunoblotting, luciferase activity, chromatin immune precipitation, electrophoretic mobility-shift assay, targeted-bisulfite-, miRNA-, RNA-, genome-sequencing, and mass-spectrometry.

Results: We found that canonical and non-canonical NF-κB signalling pathways are mandatory for A3B induction and anti-HBV effects. The degree of immune-mediated A3B production is independent of A3B promoter demethylation but is controlled post-transcriptionally by the miRNA 138-5p expression (hsa-miR-138-5p), promoting A3B mRNA decay. Hsa-miR-138-5p over-expression reduced A3B levels and its antiviral effects. Of note, established infection inhibited BS1-induced A3B expression through epigenetic modulation of A3B promoter. Twelve days of treatment with a LTβR-specific agonist BS1 is sufficient to reduce the cccDNA pool by 80% without inducing significant damages to a subset of cancer-related host genes. Interestingly, the A3B-mediated effect on HBV is independent of the transcriptional activity of cccDNA as well as on rcDNA synthesis.

Conclusions: Altogether, A3B represents the only described enzyme to target both transcriptionally active and inactive cccDNA. Thus, inhibiting hsa-miR-138-5p expression should be considered in the combinatorial design of new therapies against HBV, especially in the context of immune-mediated A3B induction.

Lay summary: Immune-mediated induction of cytidine deaminase APOBEC3B is transcriptionally regulated by NF-κB signalling and post-transcriptionally downregulated by hsa-miR-138-5p expression, leading to cccDNA decay. Timely controlled APOBEC3B-mediated cccDNA decay occurs independently of cccDNA transcriptional activity and without damage to a subset of cancer-related genes. Thus, APOBEC3B-mediated cccDNA decay could offer an efficient therapeutic alternative to target hepatitis B virus chronic infection.

Keywords: A20, tumour necrosis factor alpha-induced protein 3; APOBEC3A/A3A, apolipoprotein B mRNA editing catalytic polypeptide-like A; APOBEC3B; APOBEC3B/A3B, apolipoprotein B mRNA editing catalytic polypeptide-like B; APOBEC3G/A3G, apolipoprotein B mRNA editing catalytic polypeptide-like G; BCA, bicinchoninic acid assay; CHB, chronic hepatitis B; CXCL10, C-X-C motif chemokine ligand 10; ChIP, chromatin immune precipitation; EMSA, electrophoretic mobility-shift assay; H3K4Me3, histone 3 lysine 4 trimethylation; HBx; Hepatitis B virus; IFNα/γ, interferon alpha/gamma; IKKα/β, IκB kinase alpha/beta; JMJD8, jumonji domain containing 8; LPS, lipopolysaccharide; LTβR, lymphotoxin beta receptor; MAPK, mitogen-activated protein kinase; NEMO, NF-κB essential modulator; NF-κB; NF-κB, nuclear factor kappa B; NIK, NF-κB inducing kinase; NT, non-treated; RT-qPCR, reverse transcription-quantitative PCR; RelA, NF-κB p65 subunit; TNF, tumour necrosis factor; UBE2V1, ubiquitin conjugating enzyme E2 V1; UTR, untranslated region; cccDNA; cccDNA, covalently closed circular DNA; d.p.i., days post infection; miRNA; miRNA, micro RNA; siCTRL, siRNA control.

Graphical abstract

Fig. 1

Lymphotoxin beta receptor agonisation induces APOBEC3B through NF-κB signalling. (A) dHepaRG were treated for indicated times with 0.5 μg/ml BS1. Upper panel: schematic representation of the experiment. Lower panel: labelled probes containing the NF-κB binding sites were analysed by EMSA. (B) HEK293T cells were co-transfected with a luciferase construct containing APOBEC3B promoter (-230, +18, distance to transcription start site) wt or mutated for each NF-κB binding sites, together with NF-κB-transcription-factors-expressing plasmids. Upper panel: schematic representation of the experiment. Lower panel: schematic representation of the downstream promoter region with the inserted mutations of the NF-κB sites. Luciferase activity was assessed 48 h post-transfection. Heat map represents the mean of 1 experiment performed in triplicate. (C,D) dHepaRG were treated for the indicated time with 0.5 μg/ml BS1. Upper panel: schematic representation of the experiment. Lower panel: binding of (C) p52 and (D) polymerase II to APOBEC3B promoter was analysed by ChIP and qPCR. (E) dHepaRG were transfected with 10 nM of control or NIK-targeting siRNAs for 24 h before being left untreated (NT) or treatment with 0.5 μg/ml of BS1 ± 10 μM of TPCA-1 or 5 μM PHA-408. Upper panel: schematic representation of the experiment. Lower panel: mRNAs were isolated and analysed by RT-qPCR. (F) Knockout dHepaRG lines for NIK (sgNIK), IKKβ (sgIKKβ), NIK and IKKβ (sgNIK + sgIKKβ), RelB (sgRelB), or RelA and RelB (sgRelA + sgRelB), as well as control dHepaRG (sgCtrl) were left untreated (NT) or treated with 0.5 μg/ml of BS1 for 3 days. Upper panel: schematic representation of the experiment. Lower panel: mRNAs were isolated and analysed by RT-qPCR. Bars represent the mean ± SD of (E) 2, (F) 3, or (C,D) 4 independent experiments. Data were submitted to (C–F) 1-way ANOVA. ∗∗p <0.01; ∗∗∗p <0.001; ∗∗∗∗p <0.001. APOBEC3B, apolipoprotein B mRNA editing catalytic polypeptide-like A; ChIP, chromatin immune precipitation; EMSA, electrophoretic mobility-shift assay; IKKβ, inhibitor of nuclear factor kappa B kinase subunit beta; NF-κB, nuclear factor kappa B; NIK, NF-κB inducing kinase; n.s., not significant; wt, wild-type.

Fig. 2

APOBEC3B is post-transcriptionally regulated by the miRNA-138-5p. (A,B) Treatment of dHepaRG with 0.5 μg/ml BS1 was started sequentially and stopped altogether at the indicated time points. (A) Upper panel: schematic representation of the experiment. Lower panel: mRNAs of interest were extracted and analysed by RT-qPCR. (B) Proteins were analysed by immunoblotting. (C) Schematic representation of the working hypothesis. (D) dHepaRG were treated for 2 or 4 days with 0.5 μg/ml of BS1. Upper panel: schematic representation of the experiment. Lower panel: RNAs were extracted and small RNA were sequenced. Top 50 significantly dysregulated miRNAs of combined sequencing and RT-qPCR data was unbiased clustered and plotted. Cluster I represents miRNAs highly expressed at Day 2 and lowly expressed at Day 4 (i.e. miRNAs of interest); Cluster II represents miRNAs lowly expressed at Day 2 and highly expressed at Day 4; Cluster III represents miRNA lowly expressed at Day 2 and Day 4. (E) Schematic illustration of the miRNA-138-5p binding site on the APOBEC3B 3′-UTR. (F) dHepaRG were left untreated (NT) or treated with 0.5 μg/ml of BS1 for 2 or 4 days (see schematic representation of the experiment in D). miRNAs were extracted and analysed by RT-qPCR. (G,H) HEK293T cells were co-transfected with luciferase-3′-UTR fusion constructs and either miR-138-5p-expressing plasmids or control miR-expressing plasmids. (G) Schematic representations of luciferase-3′-UTR fusions used. (H) Upper panel: schematic representation of the experiment. Lower panel: luciferase activity was assayed 48 h post transfection. Bars, respectively points, represent the mean ± SD of (F, H) 1 experiment, or (A) 3 experiments performed in triplicate. Data were submitted to (A, F, H) unpaired Student’s t test. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; ∗∗∗∗p <0.001. APOBEC3B, apolipoprotein B mRNA editing catalytic polypeptide-like A; n.s., not significant; UTR, untranslated transcribed region.

Fig. 3

Dysregulation of APOBEC3B expression by disruption of NF-κB signalling and mi-RNA 138-5p prevent the antiviral effect. (A) Schematic representation of the experiment presented in panels B and C. (B,C) Knockout dHepaRG lines for NIK (sgNIK), IKKβ (sgIKKβ), or NIK and IKKβ (sgNIK + sgIKKβ), as well as control dHepaRG (sgCtrl) were infected with HBV. Seven-d.p.i. cells were left untreated (NT) or treated with 0.5 μg/ml of BS1 or 0.5 μM of tenofovir for 12 days. (B) DNA and (C) RNAs were isolated and analysed using RT-qPCR or qPCR. (D–G) dHepaRG were infected with HBV and 10 and 13 d.p.i. transfected with 10 nM microRNA (miR)-138-5p or control mimics. Cells were then left untreated (NT) or treated for 6 days with 0.5 μg/ml of BS1 or 0.5 μM of tenofovir. (D) Schematic representation of the experiment presented in panels E–G. (E) RNAs, (F) proteins, and (G) DNA were isolated and analysed using RT-qPCR, immunoblotting, and qPCR, respectively. Bars represent the mean ± SD of (B,C) 3 or (E, G) 6 independent experiments. Data were submitted to (B, C, E, G) 1-way ANOVA. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; ∗∗∗∗p <0.001. APOBEC3B, apolipoprotein B mRNA editing catalytic polypeptide-like A; d.p.i., days post infection; IKKβ: inhibitor of nuclear factor kappa B kinase subunit beta; NF-κB, nuclear factor kappa B; NIK, NF-κB inducing kinase; n.s., not significant.

Fig. 4

HBV-mediated inhibition of APOBEC3B expression is not sufficient to prevent the antiviral effect. (A) Schematic representation of the experiment presented in panels B–E. (B,C) dHepaRG or (D,E) PHH were infected with HBV and left untreated (NT) or treated with 0.5 μg/ml of BS1 starting 1 d.p.i, for 6 days. (B,D) RNAs were isolated and analysed using RT-qPCR or qPCR. (C,E) Levels of HBeAg were detected in the cell culture supernatant via ELISA. Bars represent the mean ± SD of (D,E) 1 experiment, or (B,C) 3 independent experiments. Data were submitted to (B-E) unpaired Student’s t test. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; ∗∗∗∗p <0.001. d.p.i., day post infection; n.s., not significant; PHH, primary human hepatocytes.

Fig. 5

APOBEC3B induction does not induce de novo mutations in a subset of genes related to cancer development. (A–E) dHepaRG were infected and 10 d.p.i. were left untreated (NT) or treated with 0.5 μg/ml BS1 for 12 days. DNA was extracted and subjected to panel sequencing of a panel containing 766 genes (CeGaT CancerPrecision panel). A total of 2,868 single nucleotide variants (SNVs) were detected. (A) Schematic representation of the experiment. (B) These SNVs were then filtered to identify SNVs, that occur in all samples with a number of alleles (NAF) >5% and a coverage >30 (2,404), only in all treated samples (13) and only in all ‘not treated’ samples (12). Four hundred and thirty-nine SNVs were not found in all samples but they were not specific to either of the 2 groups. Inspection of the 13 and 12 genes showed that they have NAFs close to the cut-off of 5% but are detected in the other samples also. (C–E) SNVs in every possible trinucleotide context were analysed for their frequency. (C) Comparison of the frequency of SNVs between non-treated and BS1 treated samples. In the table, the median frequency and the IQR of SNVs are presented. In the box plot, every data point represents a SNV in a trinucleotide context. Data were submitted to the Wilcoxon-signed Rank Sum test. (D) Frequency for all SNVs in a trinucleotide context of non-treated samples. (E) Frequency for all SNVs in a trinucleotide context of non-treated samples. APOBEC3B, apolipoprotein B mRNA editing catalytic polypeptide-like A; d.p.i., days post infection.

Fig. 6

APOBEC3B effect on double-stranded DNA is independent of transcription. (A–C) dHepaRG were infected with wild-type (wt) HBV or HBx deficient (ΔX) HBV. Seven d.p.i, cells were left untreated (NT) or treated with 0.5 μg/ml BS1 for 11 days. (A) Schematic representation of the experiment. (B,C) DNA were extracted and analysed using (B) qPCR and (C) Southern-blotting. (D–I) dHepaRG were infected with a recombinant tRFP-rHBV virus. Seven d.p.i., cells were left untreated (NT) or treated with 0.5 μg/ml BS1 or 0.5 μM of tenofovir for 9 days. (D) Schematic representation of the experiments presented in panels E–I. (E) Representative photos of bright field and fluorescent microscopy of the different treatments at 6 d.p.i. (F) Quantification of the number of RFP positive cells per view field. (G,I) RNA and (H) DNA were extracted and quantified by RT-qPCR and qPCR. Bars represent the mean ± SD of (F–I) 2 or (B) 4 independent experiments performed in triplicate. Data were submitted to (B) unpaired Student’s t test or (F–I) 1-way ANOVA. ∗∗∗p <0.001; ∗∗∗∗p <0.001. APOBEC3B, apolipoprotein B mRNA editing catalytic polypeptide-like A; d.p.i., days post infection; n.s., not significant.

Fig. 7

APOBEC3B induction and subsequent cccDNA decay depend on NF-κB signalling and miR-138-5p decrease. Graphical representation of the main proposed mechanism(s). Upon short-time agonisation of the LTβR, NF-κB signalling induces weak APOBEC3B mRNA expression because of the inhibitory activity of miR-138-5p, thereby preventing cccDNA decay. Upon a prolonged agonisation of LTβR, the miR-138-5p levels is decreased allowing potent induction of APOBEC3B mRNA, and subsequently cccDNA decay that is independent of cccDNA transcriptional activity. APOBEC3B, apolipoprotein B mRNA editing catalytic polypeptide-like A; cccDNA, covalently closed circular DNA; NF-κB, nuclear factor kappa B.