TP14: Establishment and fate of hepatitis B virus cccDNA

HBV persists by establishing a covalently closed circular (ccc) DNA form in the nucleus of infected hepatocytes, which serves as transcription template for all viral RNAs. Current antiviral treatment does not target established HBV cccDNA. In contrast, cytokines can induce modification and degradation of HBV cccDNA, but also influence its transcriptional activity. This may finally contribute to curing chronic hepatitis B. The overarching goal of this project is to understand the molecular details of the establishment of cccDNA, the maintenance of its integrity and its fate. Identified mechanisms shall serve as the basis for innovative therapies targeting HBV cccDNA.

Prof. Dr. Ulrike Protzer

Dr. Jochen Martin Wettengel

Stadler D, Kachele M, Jones AN, Hess J, Urban C, Schneider J, Xia Y, Oswald A, Nebioglu F, Bester R, Lasitschka F, Ringelhan M, Ko C, Chou WM, Geerlof A, van de Klundert MA, Wettengel JM, Schirmacher P, Heikenwalder M, Schreiner S, Bartenschlager R, Pichlmair A, Sattler M, Unger K, Protzer U. (2021) Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20. EMBO Rep. 22, e49568. doi:10.15252/embr.201949568.

Ko C, Su J, Festag J, Bester R, Kosinska AD, Protzer U. (2021) Intramolecular recombination enables the formation of hepatitis B virus (HBV) cccDNA in mice after HBV genome transfer using recombinant AAV vectors. Antiviral Res. 194, 105140. doi:10.1016/j.antiviral.2021.105140.

Wettengel JM, Linden B, Esser K, Laue M, Burwitz BJ, Protzer U. (2021) Rapid and Robust Continuous Purification of High-Titer Hepatitis B Virus for In Vitro and In Vivo Applications. Viruses. 13. doi: 10.3390/v13081503

Oswald A, Chakraborty A, Ni Y, Wettengel JM, Urban S, Protzer U. (2021) Concentration of Na(+)-taurocholate-cotransporting polypeptide expressed after in vitro-transcribed mRNA transfection determines susceptibility of hepatoma cells for hepatitis B virus. Sci Rep. 11, 19799. doi:10.1038/s41598-021-99263-3.

Esser K*, Cheng X*, Wettengel JM*, Lucifora J, Hansen-Palmus L, Austen K, Roca Suarez AA, Heintz S, Testoni B, Nebioglu F, Pham MT, Yang S, Zernecke A, Wohlleber D, Ringelhan M, Broxtermann M, Hartmann D, Huser N, Mergner J, Pichlmair A, Thasler WE, Heikenwalder M, Gasteiger G, Blutke A, Walch A, Knolle PA, Bartenschlager R, Protzer U. (2023) Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes. Cell Mol Gastroenterol Hepatol. 16, 201-221. doi: 10.1016/j.jcmgh.2023.03.011.

Ko C, Chakraborty A, Chou WM, Hasreiter J, Wettengel JM, Stadler D, Bester R, Asen T, Zhang K, Wisskirchen K, McKeating JA, Ryu WS, Protzer U. (2018) Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels. J Hepatol. 69, 1231-1241. doi:10.1016/j.jhep.2018.08.012

Lucifora J, Xia Y, Reisinger F, Zhang K, Stadler D, Cheng X, Sprinzl MF, Koppensteiner H, Makowska Z, Volz T, Remouchamps C, Chou WM, Thasler WE, Huser N, Durantel D, Liang,TJ, Munk C, Heim MH, Browning JL, Dejardin E, Dandri M, Schindler M, Heikenwalder M, Protzer U. (2014) Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science. 343, 1221-1228. doi:10.1126/science.1243462.

Xia Y, Stadler D, Lucifora J, Reisinger F, Webb D, Hosel M, Michler T, Wisskirchen K, Cheng X, Zhang K, Chou WM, Wettengel JM, Malo A, Bohne F, Hoffmann D, Eyer F, Thimme R, Falk CS, Thasler WE, Heikenwalder M, Protzer U. (2016) Interferon-gamma and Tumor Necrosis Factor-alpha Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis. Gastroenterology. 150, 194-205. doi:10.1053/j.gastro.2015.09.026.

Burwitz BJ*, Wettengel JM*, Muck-Hausl MA*, Ringelhan M, Ko C, Festag MM, Hammond KB, Northrup M, Bimber BN, Jacob T, Reed JS, Norris R, Park B, Moller-Tank S, Esser K, Greene JM, Wu HL, Abdulhaqq S, Webb G, Sutton WF, Klug A, Swanson T, Legasse AW, Vu TQ, Asokan A, Haigwood NL, Protzer U*, Sacha JB.* (2017) Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques. Nat Commun. 8, 2146. doi:10.1038/s41467-017-01953-y.

Chakraborty A, Ko C, Henning C, Lucko A, Harris JM, Chen F, Zhuang X, Wettengel JM, Roessler S, Protzer U, McKeating JA. (2020) Synchronised infection identifies early rate-limiting steps in the hepatitis B virus life cycle. Cell Microbiol. 22, e13250. doi:10.1111/cmi.13250.

Project in detail +

Receptor and intracellular protein expression determine whether HBV can establish its cccDNA in the nu-cleus. Using rHBV expressing Cre recombinase and cells that allow both positive and negative selection of infected hepatocytes, we performed a first genome-wide, pilot CRISPR knock-out screen for cellular fac-tors setting strict conditions. With this, we confirmed ZCCHC14 and identified VPS4 as a novel host cell factor determining cellular permissiveness for HBV. We will repeat the CRISPR screen in the next funding period using an alternative gRNA library and less strict conditions to obtain a more extensive and comple-mentary set of hits. In addition, in collaboration with Pichlmair (TP24N) and Z03, we will use stable-isotope labelling of proteins in cell culture (SILAC) to capture the proteome before, during, and shortly after HBV cell entry to identify differences in the protein composition between infected and non-infected cells when the virus is taken up. Novel rHBV variants expressing fast-maturing fluorescent proteins will allow the sep-aration of infected from non-infected cells. We will then confirm the role of our hits with Bartenschlager (TP09) and Z02, study the molecular mechanism together with Schreiner (TP16), and analyse together with Urban/Nkongolo (TP15) in which step of the HBV replication cycle these host cell factors are involved. Gained information will instruct the development of improved HBV infection models that allow for the screening of therapeutic approaches targeting cccDNA.
We know from our previous work that cytokines can induce either A3A or A3B that deaminate cccDNA and cause non-cytolytic purging of the episome from the nucleus of infected cells. We have also shown that degradation of cccDNA requires induction of an additional ISG, the exonuclease ISG20, coop-erating with A3A. By a proteome screen performed together with Pichlmair (TP24N), we identified an addi-tional ISG, the zink-finger protein ZAP, that also binds deaminated cccDNA and hypothesize that ZAP re-cruits the nuclease to the deaminated cccDNA. Performing an analogous approach as we have used to identify the ISG20, we will study which nuclease cooperates with A3B in NFB-mediated cccDNA reduction and aim at determining which glycosylase(s) remove the bases after deamination to target cccDNA for degradation. Together with Schreiner (TP16), Schütz/Seitz (TP23), and Bartenschlager (TP09), we thus aim to define the complete enzymatic cascade and molecular mechanism involved in modifying and finally degrading HBV cccDNA.
Based on our results that deamination can target HBV cccDNA for degradation, we will take ad-vantage of designer base editors to translate our findings into a novel therapeutic approach. We will evalu-ate their potential to target HBV cccDNA in cell culture and in vivo. Due to its lower off-target activity, we will exploit adenine instead of cytosine base-editing or use combined dual-deaminase variants with in-creased target specificity and target them preferentially to the regulatory elements within the HBV genome utilizing a set of optimized guide RNAs and functionally modified Cas9 mutants fused to the designer deam-inases.
These studies will unravel the molecular mechanisms governing cccDNA establishment, integrity, and control and lay the ground for novel approaches targeting this central reservoir of HBV persistence.