[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Faure-Dupuy, Suzanne; Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany ; Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
Riedl, Tobias ; Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany ; Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
Rolland, Maude ; Université de Liège - ULiège > GIGA > GIGA I3 - Molecular Immunology and Signal Transduction
Hizir, Zoheir ; Université de Liège - ULiège > GIGA > GIGA I3 - Molecular Immunology and Signal Transduction
Reisinger, Florian; Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany ; Institute of Virology, Helmholtz Zentrum München, Munich, Germany
Neuhaus, Katharina ; Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
Schuehle, Svenja; Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany ; Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
Remouchamps, Caroline ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Laboratory of Molecular Immunology and Signal Transduction
Gillet, Nicolas ; Université de Liège - ULiège > Agronomie, Bio-ingénierie et Chimie (AgroBioChem) > Microbial, food and biobased technologies ; Integrated Veterinary Research Unit, Namur Research Institute for Life Sciences, Namur, Belgium
Schönung, Maximilian; Faculty of Biosciences, Heidelberg University, Heidelberg, Germany ; Section Translational Cancer Epigenomics, Division of Translational Medical Oncology, German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), Heidelberg, Germany
Stadler, Mira; Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany ; Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
Wettengel, Jochen; Institute of Virology, Helmholtz Zentrum München, Munich, Germany
Barnault, Romain ; INSERM, U1052, Cancer Research Center of Lyon (CRCL), University of Lyon (UCBL1), CNRS UMR_5286, Centre Léon Bérard (CLB), Lyon, France
Parent, Romain ; INSERM, U1052, Cancer Research Center of Lyon (CRCL), University of Lyon (UCBL1), CNRS UMR_5286, Centre Léon Bérard (CLB), Lyon, France
Schuster, Linda Christina; Division of Chromatin Networks, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany
Farhat, Rayan; INSERM, U1052, Cancer Research Center of Lyon (CRCL), University of Lyon (UCBL1), CNRS UMR_5286, Centre Léon Bérard (CLB), Lyon, France
Prokosch, Sandra; Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
Leuchtenberger, Corinna; Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
Öllinger, Rupert; Institute of Molecular Oncology and Functional Genomics, Rechts der Isar University Hospital, Munich, Germany
Engleitner, Thomas ; Institute of Molecular Oncology and Functional Genomics, Rechts der Isar University Hospital, Munich, Germany
Rippe, Karsten ; Division of Chromatin Networks, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany
Rad, Roland; Institute of Molecular Oncology and Functional Genomics, Rechts der Isar University Hospital, Munich, Germany
Unger, Kristian; Research Unit of Radiation Cytogenetics, Helmholtz Zentrum München, Neuherberg, Germany
Tscharahganeh, Darjus; Helmholtz-University Group 'Cell Plasticity and Epigenetic Remodeling', German Cancer Research Center (DKFZ) and Institute of Pathology University Hospital, Heidelberg, Germany
Lipka, Daniel B; Section Translational Cancer Epigenomics, Division of Translational Medical Oncology, German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), Heidelberg, Germany ; Faculty of Medicine, Otto-von-Guericke-University, Magdeburg, Germany
Protzer, Ulrike; Institute of Virology, Helmholtz Zentrum München, Munich, Germany
Durantel, David; INSERM, U1052, Cancer Research Center of Lyon (CRCL), University of Lyon (UCBL1), CNRS UMR_5286, Centre Léon Bérard (CLB), Lyon, France
Lucifora, Julie ; INSERM, U1052, Cancer Research Center of Lyon (CRCL), University of Lyon (UCBL1), CNRS UMR_5286, Centre Léon Bérard (CLB), Lyon, France
Dejardin, Emmanuel ✱; Université de Liège - ULiège > Département des sciences biomédicales et précliniques
Heikenwälder, Mathias ✱; Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany ; Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
M.H. was supported by an ERC Consolidator grant (HepatoMetaboPath), SFBTR179 Project-ID 272983813, SFB/TR 209 Project-ID 314905040, SFBTR1335 Project-ID 360372040, the Wilhelm Sander-Stiftung, a Horizon 2020 grant (Hepcar), Research Foundation Flanders (FWO) under grant 30826052 (EOS Convention MODEL-IDI), Deutsche Krebshilfe projects 70113166 and 70113167, German-Israeli Cooperation in Cancer Research (DKFZ-MOST) and the Helmholtz-Gemeinschaft, Zukunftsthema ?Immunology and Inflammation? (ZT-0027), and the Rainer Hoenig Stiftung. E.D. and M.H. were supported by the FNRS/FWO under EOS project no. 30826052. E.D. received financial support from the F.R.S.-FNRS (CDR-J.0049.20) and the Fondation L?on Fredericq of the University of Liege. Z.H. was supported a Grant T?l?vie, Belgium. M.H., E.D., D.D., J.L., and M.R. were supported by an FP7-Infect-Era grant and a fellowship from the WBI (Wallonie-Bruxelles International). D.D. and J.L. were supported by INSERM (Institut National de la Sant? et de la Recherche M?dicale; salaries and core-fundings), ANRS (Agence Nationale de Recherche sur le Sida et les h?patites virales, several grants from study section 12 [CSS12]), and EU-Infect Era ?Target HDV? (ANR 16-IFEC-0005-01).M.H. was supported by an ERC Consolidator grant (HepatoMetaboPath), SFBTR179 Project-ID 272983813, SFB/TR 209 Project-ID 314905040, SFBTR1335 Project-ID 360372040, the Wilhelm Sander-Stiftung , a Horizon 2020 grant (Hepcar), Research Foundation Flanders (FWO) under grant 30826052 (EOS Convention MODEL-IDI), Deutsche Krebshilfe projects 70113166 and 70113167 , German-Israeli Cooperation in Cancer Research (DKFZ-MOST) and the Helmholtz-Gemeinschaft , Zukunftsthema “Immunology and Inflammation” (ZT-0027), and the Rainer Hoenig Stiftung . E.D. and M.H. were supported by the FNRS/FWO under EOS project no. 30826052. E.D. received financial support from the F.R.S.-FNRS (CDR-J.0049.20) and the Fondation Léon Fredericq of the University of Liege . Z.H. was supported a Grant Télévie , Belgium. M.H., E.D., D.D., J.L., and M.R. were supported by an FP7-Infect-Era grant and a fellowship from the WBI (Wallonie-Bruxelles International). D.D. and J.L. were supported by INSERM (Institut National de la Santé et de la Recherche Médicale; salaries and core-fundings), ANRS (Agence Nationale de Recherche sur le Sida et les hépatites virales, several grants from study section 12 [CSS12]), and EU-Infect Era “Target HDV” ( ANR 16-IFEC-0005-01 ).
Fanning, G.C., Zoulim, F., Hou, J., Bertoletti, A., Therapeutic strategies for hepatitis B virus infection: towards a cure. Nat Rev Drug Discov 18 (2019), 827–844, 10.1038/s41573-019-0037-0.
Lucifora, J., Xia, Y., Reisinger, F., Zhang, K., Stadler, D., Cheng, X., et al. Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science 343 (2014), 1221–1228, 10.1126/science.1243462.
Koh, S., Kah, J., Tham, C.Y.L., Yang, N., Ceccarello, E., Chia, A., et al. Nonlytic Lymphocytes Engineered to Express Virus-Specific T-Cell Receptors Limit HBV Infection by Activating APOBEC3. Gastroenterology 155 (2018), 180–193.e6, 10.1053/j.gastro.2018.03.027.
Xia, Y., Stadler, D., Lucifora, J., Reisinger, F., Webb, D., Hösel, M., et al. Interferon-γ and Tumor Necrosis Factor-α Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis. Gastroenterology 150 (2016), 194–205, 10.1053/j.gastro.2015.09.026.
Covino, D.A., Gauzzi, M.C., Fantuzzi, L., Understanding the regulation of APOBEC3 expression: Current evidence and much to learn. J Leukoc Biol 103 (2018), 433–444, 10.1002/JLB.2MR0717-310R.
Dejardin, E., The alternative NF-kappaB pathway from biochemistry to biology: pitfalls and promises for future drug development. Biochem Pharmacol 72 (2006), 1161–1179, 10.1016/j.bcp.2006.08.007.
Dejardin, E., Droin, N.M., Delhase, M., Haas, E., Cao, Y., Makris, C., et al. The Lymphotoxin-β Receptor Induces Different Patterns of Gene Expression via Two NF-κB Pathways. Immunity 17 (2002), 525–535, 10.1016/S1074-7613(02)00423-5.
Crispe, I.N., The liver as a lymphoid organ. Annu Rev Immunol 27 (2009), 147–163, 10.1146/annurev.immunol.021908.132629.
Wu, C.-J., Lu, L.-F., MicroRNA in Immune Regulation. Curr Top Microbiol Immunol 410 (2017), 249–267, 10.1007/82_2017_65.
Gripon, P., Rumin, S., Urban, S., Le Seyec, J., Glaise, D., Cannie, I., et al. Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A 99 (2002), 15655–15660, 10.1073/pnas.232137699.
Schulze-Bergkamen, H., Untergasser, A., Dax, A., Vogel, H., Büchler, P., Klar, E., et al. Primary human hepatocytes–a valuable tool for investigation of apoptosis and hepatitis B virus infection. J Hepatol 38 (2003), 736–744, 10.1016/s0168-8278(03)00120-x.
Namineni, S., O'Connor, T., Faure-Dupuy, S., Johansen, P., Riedl, T., Liu, K., et al. A dual role for hepatocyte-intrinsic canonical NF-κB signaling in virus control. J Hepatol, 2020, 10.1016/j.jhep.2019.12.019.
Labun, K., Montague, T.G., Gagnon, J.A., Thyme, S.B., Valen, E., CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res 44 (2016), W272–W276, 10.1093/nar/gkw398.
easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting | Nucleic Acids Research | Oxford Academic n.d. https://academic.oup.com/nar/article/44/19/e149/2468398 (accessed March 19, 2020).
Chernokalskaya, E., Dompenciel, R., Schoenberg, D.R., Cleavage properties of an estrogen-regulated polysomal ribonuclease involved in the destabilization of albumin mRNA. Nucleic Acids Res 25 (1997), 735–742, 10.1093/nar/25.4.735.
Sommers, C.D., Thompson, J.M., Guzova, J.A., Bonar, S.L., Rader, R.K., Mathialagan, S., et al. Novel tight-binding inhibitory factor-kappaB kinase (IKK-2) inhibitors demonstrate target-specific anti-inflammatory activities in cellular assays and following oral and local delivery in an in vivo model of airway inflammation. J Pharmacol Exp Ther 330 (2009), 377–388, 10.1124/jpet.108.147538.
Podolin, P.L., Callahan, J.F., Bolognese, B.J., Li, Y.H., Carlson, K., Davis, T.G., et al. Attenuation of Murine Collagen-Induced Arthritis by a Novel, Potent, Selective Small Molecule Inhibitor of IκB Kinase 2, TPCA-1 (2-[(Aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide), Occurs via Reduction of Proinflammatory Cytokines and Antigen-Induced T Cell Proliferation. J Pharmacol Exp Ther 312 (2005), 373–381, 10.1124/jpet.104.074484.
Riedl, T., Faure-Dupuy, S., Rolland, M., Schuehle, S., Hizir, Z., Calderazzo, S., et al. HIF1α-mediated RelB/APOBEC3B downregulation allows Hepatitis B Virus persistence. Hepatol Baltim Md 74:4 (2021 oct), 1766–1781, 10.1002/hep.31902 Epub 2021 Aug 15.
Schönung, M., Hess, J., Bawidamann, P., Stäble, S., Hey, J., Langstein, J., et al. AmpliconDesign – an interactive web server for the design of high-throughput targeted DNA methylation assays. Epigenetics 16:9 (2021 Sep), 933–939, 10.1080/15592294.2020.1834921 Epub 2020 Oct 24.
Burns, M.B., Lackey, L., Carpenter, M.A., Rathore, A., Land, A.M., Leonard, B., et al. APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494 (2013), 366–370, 10.1038/nature11881.
Chen, Y., Hu, J., Cai, X., Huang, Y., Zhou, X., Tu, Z., et al. APOBEC3B edits HBV DNA and inhibits HBV replication during reverse transcription. Antiviral Res 149 (2018), 16–25, 10.1016/j.antiviral.2017.11.006.
McDaniel, Y.Z., Wang, D., Love, R.P., Adolph, M.B., Mohammadzadeh, N., Chelico, L., et al. Deamination hotspots among APOBEC3 family members are defined by both target site sequence context and ssDNA secondary structure. Nucleic Acids Res 48 (2020), 1353–1371, 10.1093/nar/gkz1164.
Lucifora, J., Arzberger, S., Durantel, D., Belloni, L., Strubin, M., Levrero, M., et al. Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection. J Hepatol 55 (2011), 996–1003, 10.1016/j.jhep.2011.02.015.
Belloni, L., Pollicino, T., De Nicola, F., Guerrieri, F., Raffa, G., Fanciulli, M., et al. Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proc Natl Acad Sci U S A 106 (2009), 19975–19979, 10.1073/pnas.0908365106.
Doehle, B.P., Schäfer, A., Cullen, B.R., Human APOBEC3B is a potent inhibitor of HIV-1 infectivity and is resistant to HIV-1 Vif. Virology 339 (2005), 281–288, 10.1016/j.virol.2005.06.005.
Warren, C.J., Westrich, J.A., Van Doorslaer, K., Pyeon, D., Roles of APOBEC3A and APOBEC3B in Human Papillomavirus Infection and Disease Progression. Viruses, 9, 2017, 10.3390/v9080233.
Maruyama, W., Shirakawa, K., Matsui, H., Matsumoto, T., Yamazaki, H., Sarca, A.D., et al. Classical NF-κB pathway is responsible for APOBEC3B expression in cancer cells. Biochem Biophys Res Commun 478 (2016), 1466–1471, 10.1016/j.bbrc.2016.08.148.
Cao, W., Wu, W., MicroRNAs regulate APOBEC gene expression. Histol Histopathol 33 (2018), 117–120, 10.14670/HH-11-912.
Yeh, M., Oh, C.S., Yoo, J.Y., Kaur, B., Lee, T.J., Pivotal role of microRNA-138 in human cancers. Am J Cancer Res 9 (2019), 1118–1126.
Luangsay, S., Gruffaz, M., Isorce, N., Testoni, B., Michelet, M., Faure-Dupuy, S., et al. Early inhibition of hepatocyte innate responses by hepatitis B virus. J Hepatol 63 (2015), 1314–1322, 10.1016/j.jhep.2015.07.014.
Liu, M., Mallinger, A., Tortorici, M., Newbatt, Y., Richards, M., Mirza, A., et al. Evaluation of APOBEC3B Recognition Motifs by NMR Reveals Preferred Substrates. ACS Chem Biol 13 (2018), 2427–2432, 10.1021/acschembio.8b00639.
Siriwardena, S.U., Chen, K., Bhagwat, A.S., Functions and Malfunctions of Mammalian DNA-Cytosine Deaminases. Chem Rev 116 (2016), 12688–12710, 10.1021/acs.chemrev.6b00296.
Tu, T., Budzinska, M.A., Shackel, N.A., Urban, S., HBV DNA Integration: Molecular Mechanisms and Clinical Implications. Viruses, 9, 2017, 10.3390/v9040075.
