HBV persists by establishing a covalently closed circular (ccc) DNA form in the nucleus of infected hepato-cytes, which serves as transcription template for all viral RNAs. Antiviral treatment with available nucle-os(t)ide analogues or with entry or capsid assembly inhibitors in development cannot directly affect or di-minish established HBV cccDNA. Modification as well as degradation influence activity and total levels of HBV cccDNA, and are regarded to form the basis for the development of new treatment approaches that for the first time would allow direct targeting of HBV cccDNA. This may then finally allow curing hepatitis B. The overarching goal of this project is to understand cccDNA establishment, integrity and control in molecular detail to allow therapeutic targeting of cccDNA which is the holy grail in “HBV Cure” re-search.
We have demonstrated that nuclear HBV cccDNA is specifically degraded by cellular endonucleases after it has been modified by interferon - or lymphotoxin-induced, nuclear APOBEC3 (A3) family deaminases A3A and A3B, respectively (Lucifora, Xia et al. 2014), or by T cell cytokines (Xia, Stadler et al. 2016). Most recent-ly we could show that type I, II and III interferons activate the same pathway (Bockmann et al. 2019) and obtained first evidence that it is also active in patients’ livers after interferon therapy (Li, Xia et al. 2017). We demonstrated that overexpression of A3A and A3B are essential, but not sufficient to induce cccDNA deg-radation, and that cccDNA degradation requires an additional interferon-stimulated gene (ISG), ISG20, acting as an exonuclease (Stadler et al., unpublished). This clearly demonstrates that ISGs cooperate in con-trolling cccDNA.
For an improved therapeutic targeting, we developed a novel, “PASylated” interferon with liver targeting capacity and an increased half-life, and found that this is able to stimulate not only innate, but also adaptive immunity and control HBV in HBV-transgenic mice (Xia et al 2019). Using liver-humanized mice infected with HBV we confirmed in vivo that primarily cytotoxic T cell function, but also T-cell derived cytokines contribute to HBV clearance (Wisskirchen et al., 2019). To improve the basis for studies on HBV cccDNA, we have intensively worked on optimizing cell culture and in vivo models of HBV infection (Ko et al. 2018; Burwitz et al. 2017) that now allow us to study cccDNA kinetics and identify factors essential for cccDNA establish-ment and maintenance taking advantage of these models and collaborations within the TRR with Heikenwälder, Schreiner, Urban and Binder/Pichlmair.
In the next funding period we plan to complete identification of the pathway(s) determining cccDNA fate and use our know-how to target cccDNA in vivo by activating the pathways involved in non-cytolytic degra-dation of cccDNA. In addition, we will define factors essential for cccDNA establishment taking advantage of data sets and reporter viruses we have produced. The project aims in the second funding period are: (1) To determine the molecular pathways controlling cccDNA fate. Here, we will continue our efforts to iden-tify all players involved in non-cytolytic degradation of cccDNA, determine the role of a transcription bubble to allow cccDNA targeting, study the involvement of HBx and study whether and how Zincfinger antiviral protein (ZAP) identified by a proteome screen controls HBV (in collaboration with Heikenwälder, Schreiner, Seitz / Schütz and Pichlmair and with Z03N). (2) To target pathway(s) involved in non-cytolytic degradation of cccDNA in vivo. Here, we will use liver-directed interferons or Rig-I ligands to induce cytokines and target cccDNA in the mouse liver and will exploit CRISPR deaminases to define which level of deamination is re-quired to render cccDNA prone to degradation (in collaboration with Grimm, Schreiner, Heikenwälder, Knolle, Wohlleber and with Z02). (3) To define factors essential for establishment of cccDNA. Here, we will perform gain-of-function and loss-of-function experiments exploiting models and HBV reporter viruses we have es-tablished to determine critical steps and proteins in cccDNA establishment. This will be complemented by an in silico analysis of proteome data we have obtained (in collaboration with Binder/Pichlmair, Urban, Bar-tenschlager, Grimm, Schreiner, Schütz/Seitz and with Z02 and Z03N)

Milestones of the first funding period.
- Determining the pathway(s) involved in non-cytolytic purging of cccDNA
- Determining the specificity of cccDNA deamination
- Determine the role of T-cell derived cytokines in purging cccDNA in vivo