TP14: Means of modification and degradation 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. Cytokines induce modification and degradation of HBV cccDNA and influence its activity. 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” research.

Prof. Dr. Ulrike Protzer

Allweiss L, Testoni B, Yu M, Lucifora J, Ko C, Qu B, Lütgehetmann M, Guo H, Urban S, Fletcher SP, Protzer U, Levrero M, Zoulim F, Dandri M. (2023) Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure. Gut. 72(5):972-983. doi: 10.1136/gutjnl-2022-328380.

Hofmann S, Plank V, Groitl P, Skvorc N, Hofmann K, Luther J, Ko C, Zimmerman P, Bruss V, Stadler D, Carpentier A, Rezk S, Nassal M, Protzer U, Schreiner S. (2023) SUMO Modification of Hepatitis B Virus Core Mediates Nuclear Entry, Promyelocytic Leukemia Nuclear Body Association, and Efficient Formation of Covalently Closed Circular DNA. Microbiol Spectr. 18:e0044623. doi: 10.1128/spectrum.00446-23.

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, Hüser 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,Cellular and Molecular Gastroenterology and Hepatology, 2023,ISSN 2352-345X,https://doi.org/10.1016/j.jcmgh.2023.03.011.

Tu T, Zehnder B, Wettengel JM, Zhang H, Coulter S, Ho V, Douglas MW, Protzer U, George J, Urban S. (2022) Mitosis of hepatitis B virus-infected cells in vitro results in uninfected daughter cells. JHEP Rep. 15;4(9):100514. doi: 10.1016/j.jhepr.2022.100514.

Kim ES, Zhou J, Zhang H, Marchetti A, van de Klundert M, Cai D, Yu X, Mitra B, Liu Y, Wang M, Protzer U, Guo H. (2022) Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA. PLoS Pathog. 9;18(6):e1010576. doi: 10.1371/journal.ppat.1010576.

Wratil PR, Stern M, Priller A, Willmann A, Almanzar G, Vogel E, Feuerherd M, Cheng CC, Yazici S, Christa C, Jeske S, Lupoli G, Vogt T, Albanese M, Mejías-Pérez E, Bauernfried S, Graf N, Mijocevic H, Vu M, Tinnefeld K, Wettengel J, Hoffmann D, Muenchhoff M, Daechert C, Mairhofer H, Krebs S, Fingerle V, Graf A, Steininger P, Blum H, Hornung V, Liebl B, Überla K, Prelog M, Knolle P, Keppler OT, Protzer U. (2022) Three exposures to the spike protein of SARS-CoV-2 by either infection or vaccination elicit superior neutralizing immunity to all variants of concern. Nat Med. 28(3):496-503. doi: 10.1038/s41591-022-01715-4.

Koerber N, Priller A , Yazici S, Bauer T, Cheng CC, Mijočević H, Wintersteller H, Jeske S, Vogel E, Feuerherd M, Tinnefeld K, Winter C, Ruland J, Gerhard M, Haller B, Christa C, Zelger O, Roggendorf H, Halle M, Erber J, Lingor P, Keppler O, Zehn D, Protzer U, Knolle PA. (2022) Dynamics of spike-and nucleocapsid specific immunity during long-term follow-up and vaccination of SARS-CoV-2 convalescents. Nat Commun 13, 153. https://doi.org/10.1038/s41467-021-27649-y.

Bunse T, Kosinska AD, Michler T, Protzer U. (2022) PD-L1 silencing in liver using siRNAs enhances efficacy of therapeutic vaccination for chronic Hepatitis B. Biomolecules. 18;12(3):470. doi: 10.3390/biom12030470.

Wagner KI, Mateyka LM, Jarosch S, Grass V, Weber S, Schober K, Hammel M, Burrell T, Kalali B, Poppert H, Beyer H, Schambeck S, Holdenrieder S, Strötges-Achatz A, Haselmann V, Neumaier M, Erber J, Priller A, Yazici S, Roggendorf H, Odendahl M, Tonn T, Dick A, Witter K, Mijočević H, Protzer U, Knolle PA, Pichlmair A, Crowell CS, Gerhard M, D'Ippolito E, Busch DH. (2022) Recruitment of highly cytotoxic CD8+ T cell receptors in mild SARS-CoV-2 infection. Cell Rep. 11;38(2):110214. doi: 10.1016/j.celrep.2021.110214.

Altstetter SM, Quitt O, Pinci F, Hornung V, Lucko A.M, Wisskirchen K, Jung S, Protzer U. (2021) Hepatitis-D virus infection is not impaired by innate immunity but increases cytotoxic T-Cell activity cells. 10(11): 3253. doi: 10.3390/cells10113253.

Knolle PA, Huang LR, Kosinska A, Wohlleber D, Protzer U. (2021) Improving therapeutic vaccination against Hepatitis B-insights from preclinical models of immune therapy against persistent Hepatitis B virus infection. Vaccines (Basel). 16;9(11):1333. doi: 10.3390/vaccines9111333.

Quitt O, Luo S, Meyer M, Xie Z, Golsaz-Shirazi F, Loffredo-Verde E, Festag J, Bockmann JH, Zhao L, Stadler D, Chou WM, Tedjokusumo R, Wettengel JM, Ko C, Noeßner E, Bulbuc N, Shokri F, Lüttgau S, Heikenwälder M, Bohne F, Moldenhauer G, Momburg F, Protzer U. (2021) T-cell engager antibodies enable T cells to control HBV infection and to target HBsAg-positive hepatoma in mice. J Hepatol. 75(5):1058-1071. doi: 10.1016/j.jhep.2021.06.022.

Faure-Dupuy S, Riedl T, Rolland M, Hizir Z, Reisinger F, Neuhaus K, Schuehle S, Remouchamps C, Gillet N, Schönung M, Stadler M, Wettengel J, Barnault R, Parent R, Schuster LC, Farhat R, Prokosch S, Leuchtenberger C, Öllinger R, Engleitner T, Rippe K, Rad R, Unger K, Tschaharganeh D, Lipka DB, Protzer U, Durantel D, Lucifora J, Dejardin E, Heikenwälder M. (2021) Control of APOBEC3B induction and cccDNA decay by NF-κB and miR-138-5p. JHEP Rep. 25;3(6):100354. doi: 10.1016/j.jhepr.2021.100354.

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. 5;11(1):19799. doi: 10.1038/s41598-021-99263-3.

Kosinska AD, Festag J, Mück-Häusl M, Festag MM, Asen T, Protzer U. (2021) Immunogenicity and antiviral response of therapeutic Hepatitis B vaccination in a mouse model of HBeAg-negative, persistent HBV infection. Vaccines (Basel). 9(8): 841. doi: 10.3390/vaccines908084.

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(8): 1503. doi: 10.3390/v13081503.

Stadler D, Kächele 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, Heikenwälder 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. 4;22(6):e49568. doi: 10.15252/embr.201949568.

Stukalov A, Girault V, Grass V, Karayel O, Bergant V, Urban C, Haas DA, Huang Y, Oubraham L, Wang A, Hamad MS, Piras A, Hansen FM, Tanzer MC, Paron I, Zinzula L, Engleitner T, Reinecke M, Lavacca TM, Ehmann R, Wölfel R, Jores J, Kuster B, Protzer U, Rad R, Ziebuhr J, Thiel V, Scaturro P, Mann M, Pichlmair A. (2021) Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV. Nature. 594(7862):246-252. doi: 10.1038/s41586-021-03493-4.

Riedl T, Faure-Dupuy S, Rolland M, Schuehle S, Hizir Z, Calderazzo S, Zhuang X, Wettengel J, Lopez MA, Barnault R, Mirakaj V, Prokosch S, Heide D, Leuchtenbergeg C, Schneider M, Heßling B, Stottmeier B, Wessbecher IM, Schirmacher P, McKeating JA, Protzer U, Durantel D, Lucifora J, Dejardin E, Heikenwalder M. (2021) Hepatology. 74(4):1766-1781. doi: 10.1002/hep.31902.

Jung S, Jacobs K, Shein M, Schütz AK, Mohr F, Stadler H, Stadler D, Lucko AM, Altstetter SM, Wilsch F, Deng L, Protzer U. (2020). Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles. J Extracell Vesicles. 10(2), e12040. https://doi.org/10.1002/jev2.12040

Namineni S, O'Connor T, Faure-Dupuy S, Johansen P, Riedl T, Liu K, Xu H, Singh I, Shinde P, Li F, Pandyra A, Sharma P, Ringelhan M, Muschaweckh A, Borst K, Blank P, Lampl S, Durantel D, Farhat R, Weber A, Lenggenhager D, Kündig TM, Staeheli P, Protzer U, Wohlleber D, Holzmann B, Binder M, Breuhahn K, Assmus LM, Nattermann J, Abdullah Z, Rolland M, Dejardin E, Lang PA, Lang KS, Karin M, Lucifora J, Kalinke U, Knolle PA, Heikenwalder M. 2020. A dual role for hepatocyte-intrinsic canonical NF-κB signaling in virus control. J Hepatol. 72(5):960-975. (TP05, TP06, TP11, TP13, TP14)

Wisskirchen K*, Kah J*, Malo A, Asen T, Volz T, Allweiss L, Wettengel JM, Lütgehetmann M, Urban S, Bauer T, Dandri M*, Protzer U*. 2019. T-Cell Receptor Grafting allows Virological Control of Hepatitis B Virus Infection. J Clin Invest. 129(7):2932-2945. * equal contribution.

Xia Y, Schlapschy M, Morath⁠ V, Roeder N⁠, Vogt EI, Stadler D⁠, Cheng X⁠⁠, Dittmer U⁠, Sutter K⁠, Heikenwalder M⁠, Skerra A⁠, Protzer U. 2019. PASylated interferon α efficiently suppresses hepatitis B virus and induces anti-HBs seroconversion in HBV-transgenic mice. Antiviral Research. 161:134-143

Bockmann JH, Stadler D, Xia Y, Ko C, Wettengel JM, Schulze zur Wiesch J, Dandri M, Protzer U. 2019. Compar-ative analysis of the antiviral effects mediated by type I and III interferons in hepatitis B virus infected hepato-cytes. J Inf Dis 220(4):567-577

Wing P, Davenne T, Wettengel J, Lai AG, Zhuang X, Chakraborty A, D’Arienzo V, Kramer C, Ko C, Harris LM, Schreiner S, Higgs M, Roessler S, Parish JL, Protzer U, Balfe P, Rehwinkel J, McKeating J.2019. A dual role for SAMHD1 in regulating HBV cccDNA and RT-dependent particle genesis. Life Sci Alliance. 2(2).

Manske K, Kallin N, König V, Schneider A, Kurz S, Bosch M, Welz M, Cheng RL, Bengsch B, Steiger K, Protzer U, Thimme R, Knolle PA, Wohlleber D. 2018. Outcome of anti-viral immunity in the liver is shaped by the level of an-tigen expressed in infected hepatocytes. Hepatology. 8(6):2089-2105.

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(6):1231-1241.

Burwitz B*, Wettengel J*, Mück-Häusl M, Ringelhan M, Ko C, Festag MM, Hammond KB, Northrup M, Bimber BN, Jacob T, Reed JS, Reed N, 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 J*. 2017. Hepatocytic Expression of Human Sodium-Taurocholate Cotransporting Polypeptide Enables Hepatitis B Virus Infection of Macaques. Nat Commun. 8(1): 2146. * equal contribution.

Li Y*, Xia Y*, Han M, Chen G, Zhang D, Thasler W, Protzer U*, Ning Q*. 2017. IFN-α-mediated Base Excision Repair Pathway Correlates with Antiviral Response Against HBV Infection. Sci Rep. 7(1):12715. * equal contribu-tion.

Xia Y*, Stadler D*, Lucifora J, Reisinger F, Webb D, Hösel M, Michler T, Wisskirchen K, Cheng X, Zhang K, Chao W-M, Wettengel J, Malo A, Bohne F, Hoffmann D, Eyer F, Thimme R, Thasler WE, Heikenwalder M, Protzer U. 2016. Interferon-γ and Tumor Necrosis Factor-α Produced by T cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis. Gastroenterology. 150(1):194-202.

Lucifora J*, Xia Y*, Reisinger F, Zhang K, Stadler D, Cheng X, Sprinzl M, Koppensteiner H, Li Y, Makowska Z, Volz T, Remouchamps C, Chou W-M, Thasler W, Hüser N, Durantel D, Münk C, Heim MH, Browning JL, Dejardin E, Dandri M, Schindler M, Heikenwalder M*, Protzer U*. 2014. Specific and non-hepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science 343(6176):1121-1128. * equal contribution.

Project in detail +

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