Interferons Induce Degradation Of HBV CccDNA
Yuchen Xia1, Julie Lucifora1, Ke Zhang1, Xiaoming cheng1, Daniela Stadler1, Florian Reisinger1, Martin Feuerherd1, Zuzanna Makowska2, Daniel Hartmann3, Wolfgang E. Thasler4, Markus H. Heim2, Mathias Heikenwälder1, Ulrike Protzer1
1lnstitute of Virology, Technische Universität München / Helmholtz Zenfrum München, Munich, Germany; 2Department of Biomedicine, University Hospital Basel, Basel, Switzerland; 3Department of Surgery University Hospital rechts der Isar, Technische Universität München, Munich, Germany; 4Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich, Germany
Persistence of HBV cccDNA in infected hepatocytes is a major problem in chronic hepatitis B treatment. Noncytopathic viral clearance by interferon (IFN) has been described, but the mechanisms involved remain elusive. In our study, we investigated if IFNs can exert degradation of HBV cccDNA, the template of HBV transcription. In HBV infected primary human hepatocytes and HepaRG cells, treatment with IFN-α and IFN-۷ significantly reduced HBV cccDNA. HBV cccDNA specific 3D-PCR indicated sequence alterations. Sequence analysis showed Cto U transition of the HBV cccDNA minus strand after IFN-α and IFN-۷ treatment. A detailed analysis of the underlying mechanism after IFN-α treatment revealed upregulation of the APOBEc3 (A3) family cytidine deaminases A3A and A3G in both cell types and in IFN-α treated patient livers in a time and dose dependent manner. JAK-STAT signaling blockade or HIV-Vif expression proved that IFN-α induced cccDNA deamination by A3 lead to degradation. Subcellllular localization analysis and overexpression experiments demonstrated that A3A, which locates to the nucleus, was the active effector. Treatment of cccDNA with a DNA repair enzyme cocktail corrected all mutations indicating that uracil could be removed by uracil-DNA glycosylase inducing apurinic/apyrimidinic (AP) sites. AP endonuclease reduced cccDNA levels in IFN-a treated cells showing that the cccDNA can be further digested by this endonuclease. We did not observe any deamination of host genomic DNA upon IFN-a treatment by 3D-PCR analysis or deep sequencing. This suggested that A3A acts on and is directed specifically to viral DNA. Since A3A co-localized with HBV core protein (HBc) in confocal microscopy and interaction was confirmed by co-immunoprecipitation, we propose that A3A utilizes HBc to get access to cccDNA. Chromatin immunoprecipitation confirmed that both HBc and A3A were bound to the cccDNA minichromosome. In HBV(x-) infection, reduction of cccDNA by IFN-α depended on trans-complementation with HBx, which is required to activate cccDNA transcription and HBc expression. Since IFN-α needs to be applied at high doses to clear infection, we screened for other cytokines showing similar antiviral effects. Like IFN-α and IFN-γ, TNF-α and more importantly activation of the lymphotoxin-β receptor at therapeutic doses were able to trigger deamination and subsequent degradation of HBV cccDNA via base excision pathway in an NF-kB dependent fashion. Our studies for the first time show that HBV cccDNA can be degraded without affecting the host cell and thus open new options for the development of novel and safe treatments to eradicate HBV and cure chronic hepatitis B.
Ulrike Protzer - Consulting: GILEAD; Grant/Research Support: Janssen
The following people have nothing to disclose: Yuchen Xia, Julie Lucifora, Ke Zhang, Xiaoming Cheng, Daniela Stadler, Florian Reisinger, Martin Feuerherd, Zuzanna Makowska, Daniel Hartmann, Wolfgang E. Thasler, Markus H. Heim, Mathias Heikenwälder