Despite the availability of an effective vaccine, around 257 million people worldwide are chronically infected with HBV. At present there is no cure because current therapies do not target the persistence reservoir of the virus, i.e. the episomal covalently closed circular DNA (cccDNA) of the HBV genome. This cccDNA is generated from the virion-borne polymerase-linked relaxed circular DNA (rcDNA) in a process that probably involves various components of the host cell DNA damage response (DDR), although the mechanism is far from being understood. We found that host SUMOylation pathways and dynamic changes in the composi-tion of promyelocytic leukemia protein (PML) nuclear bodies (PML-NBs) appear to represent a so far un-known link between DDR and cccDNA formation. We have first evidence that cccDNA conversion occurs at specific sites of SUMO conjugation at the nuclear PML-NBs with so far unknown composition. PML-NBs are interferon (IFN)-induced, multiprotein complexes with a highly dynamic composition and a role in biological functions such as DDR, apoptosis, differentiation processes and tumorigenesis. Our ongoing efforts with Binder/Pichlmair using human adenoviruses (HAdV) and adenoviral early proteins as a tool to block or ad-just host DDR during HBV infection identified several DDR factor candidates that might be regulating cccDNA establishment. In the proposed next funding period we will continue along the concept that rcDNA with its unusual molecular features is recognized by the cell as damaged DNA that evokes a DDR in the hepatocyte and evaluate the identified candidates concerning their contributions to rc to cccDNA conver-sion in liver cell knock-down systems. To understand the role of PML-associated constitutive transcription factors in regulating the HBV persistence reservoir, i.e. cccDNA, we will study Daxx/ATRX/ DEK/HIRA/H3.3 (histone acetylation), Sp100A/B/C/HMG (histone methylation), PML isoforms I-VI (PML-NB scaffold) and SUMO1-3 (PML-NB structure) in hepatocytes. In addition, we will also im¬plement more physiological hepatic models into our toolbox, in particular primary human hepatocytes (PHHs) with Protzer; stem-cell derived hepatocyte-like cells (HLC) with Dao Thi/Boulant; and spheroid cultures of liver cell lines with Heikenwälder and Binder/Pichlmair. We found that cccDNA-associated HBV proteins, i.e. HBx and HBV core (HBc) (i) represent novel targets of the host SUMOylation machinery, (ii) localize to PML-NB sites, presumably to-gether with cccDNA, and (iii) interact with specific PML constituent components. Using SUMOylation-deficient mutants of HBc we showed that SUMOylation is a prerequisite for association with specific, highly dynamic PML-NBs and generation of cccDNA from the rcDNA precursor. Thus, in the second funding peri-od, we will focus on the hypothesis that PML-NBs represent the sites of cccDNA establishment and mainte-nance. We will also analyse the longevity of cccDNA at sites of PML-NBs, as we know from HAdV replica-tion centers that viral genomes are not degraded by the host proteolysis, when associated with DDR pro-teins. To address this further, we will also study the effects of SUMO moieties and PML-NB modulation on the IFN sensing (with Protzer and Hofmann) and ISG pattern (with Binder/Pichlmair, Protzer, Urban, Barten-schlager) in HBV infected hepatocytes. We will further advance the set-up of a live-cell imaging CRISPR/dCAS system to monitor cccDNA and host PML/SUMO/DDR factors synchronously by gRNA mul-tiplexing in hepatocyte culture systems. In this way we aim to decipher cellular and viral strategies involved in the establishment and maintenance of HBV cccDNA reservoirs in the nucleus. As long-term goal, we aim to identify DDR protein candidates as promising targets for antiviral intervention strategies against chronic HBV infections.

Milestones of the first funding period:
- Development of a Cas9 expressing HepG2 master cell line
- Examine the molecular mechanism of Ad early protein abrogation of RC- to cccDNA conversion
- Elucidating the role of host DNA ligases in the final step of cccDNA formation
- Impact of the cellular DNA damage response on HBV replication and vice versa