TP16: Determinants, dynamics and diversity of SUMO/PML nuclear bodies in the control of hepatitis B virus infection and cccDNA

We will examine the role of posttranslational SUMO modification of HBV proteins, particularly core and HBx, and understand the role of this modification in the viral replication cycle. We will also study the role of HBx SUMOylation in proteasomal degradation of SMC5/6 via the DDB1/CUL4 E3 ubiquitin ligase complex and the implications for epigenetic regulation and transcription of cccDNA. Since not all PML nuclear bodies (NBs) contribute to cccDNA establishment, the goal is to uncover the precise composition of cccPML NBs. Since these nuclear structures might represent the sites of cccDNA synthesis, we will use our results to devise novel therapeutic approaches.

Prof. Dr. Sabrina Schreiner

Hofmann S, Plank V, Groitl P, Skvorc N, Hofmann K, Ko C, Zimmermann P, Bruss V, Stadler D, Carpentier A, Rezk S, Nassal M, Protzer U, Schreiner S. (2023) SUMO modification of HBV core mediates nuclear entry, PML association and efficient cccDNA formation. Microbiol Spectr. (2023) e0044623.doi:10.1128/spectrum.00446-23.

Stubbe M, Mai J, Paulus C, Stubbe HC, Berscheminski J, Karimi M, Hofmann S, Weber E, Hadian K, Hay R, Groitl P, Nevels M, Dobner T, Schreiner S. (2020) Viral DNA Binding Protein SUMOylation Promotes PML Nuclear Body Localization Next to Viral Replication Centers. mBio. 11. doi:10.1128/mBio.00049-20.

Schreiner S, Wimmer P, Dobner T. (2012) Adenovirus degradation of cellular proteins. Future Microbiol. 7(2), 211-225. doi: 10.2217/fmb.11.153.

Stadler D, Kächele M, Heß J, Schneider J, Xia Y, Lasitschka F, Ringelhan M, Ko C, Chou W, Wettengel J, Schreiner S, Pichlmair A, Schirmacher P, Heikenwälder M, Unger C, Protzer U. (2019) Identification of ISG20 as the nuclease involved in interferon-induced decline of hepatitis B virus cccDNA. EMBO Rep. (2021). doi:10.15252/embr.201949568.

Gottig L, Weiss C, Stubbe M, Hanrieder L, Hofmann S, Grodziecki A, Stadler D, Carpentier A, Protzer U, Schreiner S. (2023) Apobec3A Deamination Functions Are Involved in Antagonizing Efficient Human Adenovirus Replication and Gene Expression. mBio. 14, e0347822. doi:10.1128/mbio.03478-22.

Mai J, Stubbe M, Hofmann S, Masser S, Dobner T, Boutell C, Groitl P, Schreiner S. (2022) PML alternative splice products differentially regulate HAdV productive infection. Microbiol Spectr. e0078522.doi:10.1123/spectrum.00785-22.

Hofmann S, Mai J, Masser S, Groitl P, Hermann A, Brack-Werner R, Sternsdorf T, Schreiner S. (2020) ATO (Arsenic Trioxide) effects on PML nuclear bodies reveals antiviral intervention capacity. Adv Sci. 7(8):1902130. doi: 10.1002/advs.201902130.

Lampl S, Janas M, Donakonda S, Brugger M, Lohr K, Schneider A, Manske K, Sperl L, Wettmarshausen J, Müller C, Laschinger M, Hartmann D, Hüser N, Perrochi F, Schmitt-Kopplin P, Hagn F, Zender L, Hornung, Borner C, Pichlmair A, Kashkar H, Klingenspor M, Prinz M, Schreiner S, Conrad M, Jost P, Zischka H, Steiger K, Krönke M, Zehn D, Protzer P, Heikenwälder M, Knolle PA, Wohlleber D. (2020) Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signalling in virus infected hepatocytes. J Hepatol. 73(6), pp. 1347-1359. doi: 10.1016/j.jhep.2020.06.026.

Wing P, Davenne T, Wettengel J, Lai A, Chakraborty A, D’Arienzo V, Kramer C, Ko C, Harris J, Schreiner S, Higgs M, Parish J, Protzer U, Balfe P, Rehwinkel J, McKeating JA. (2019) A dual role for SAMHD1 in HBV cccDNA synthesis and RT-dependent particle genesis. Life Sci Alliance. 2(2):e201900355. doi: 10.26508/lsa.201900355.

Schreiner S, Nassal M. (2017) A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation- and Beyond? Viruses. 9(5):125. doi: 10.3390/v9050125 Perspective Review.

Project in detail +

Currently established therapies fail to tackle and eradicate the persistent hepatitis B virus (HBV) covalently closed circular (ccc)DNA reservoir. Thus, novel therapeutic approaches for curative treatment are urgently needed. Promyelocytic leukemia (PML) nuclear bodies (NBs) are nuclear matrix-associated multiprotein complexes that act as cellular hotspots of regulative posttranslational modification (PTM) of proteins with the small ubiquitin related modifier (SUMO). In the first funding period, we have observed that PML-NBs play a pivotal role during HBV infection based on the fact that DNA repair proteins and cccDNA synthesis are functionally connected with SUMOylation, with both being recruited to sites of PML-NBs. By using hu-man adenoviruses (HAdV) as a tool to manipulate DNA repair proteins required for cccDNA establishment, we showed that Apobec3A deaminases reported by Protzer to antagonize cccDNA levels, are regulat-ed via SUMO and PML crosstalk (with Nassal, Protzer, Binder/Pichlmair). During the ongoing funding period we provide evidence that SUMOylated HBV core protein monomers within mature HBV capsids in-duce capsid disruption at the nuclear pore complex. The SUMOylated HBV core fraction is then recruited to specific PML-NBs to promote relaxed circular (rc)- to cccDNA conversion. Thus, viral SUMOylation events and cccDNA-specific PML-NBs (cccPML-NBs) represent potential new targets to efficiently combat the cccDNA reservoir. Building on these achievements, in the next funding period we will pursue three complementary aims. In AIM1, we will biochemically analyse the covalent SUMO modification on HBV core and HBx proteins. For both HBV proteins we already know from our work performed within TRR179 that SUMOylation modulates protein function and promotes cccDNA synthesis. Therefore, we will further vali-date the chemical nature of the covalent SUMO bond via in vitro SUMOylation approaches including re-combinant proteins without the viral/host background. We will use mass spectrometry (with Pichlmair) and footprinting analysis (with Schütz/Seitz) to determine the exact nature of the SUMO modification, the exact SUMO moieties, SUMO chain formation and SUMO conjugation motif usage on both viral proteins. Previ-ously observed effects of HBV core SUMOylation on virion disassembly during entry will also be validated in vitro and via electron microscopy with recombinant virus particles and our SUMO conjugation-deficient mutants (with Schütz/Seitz and Bartenschlager). We will further dissect the role of HBx SUMOylation in the context of the DDB1/CUL4 E3 ubiquitin ligase function and chromatin remodelling via the HBx SUMOy-lation-dependent inhibition of SMC5/6. As we observed in the current funding period that not every PML-NB is supportive of cccDNA establishment, as part of AIM2 we will unravel the exact protein composition of those NBs representing the sites of cccDNA synthesis (cccPML-NBs) and the molecular microenvironment of those foci with which HBV core and HBx are associated. We will optimize imaging techniques (with Bartenschlager) to monitor cccDNA within the infected cell using FISH, CLICK chemistry, live-cell imaging (CRISPR-dCAS multiplexing), APEX and CASPEX proximity labeling and single cell mass spectrometry (with Knolle, Pichlmair, Z03) to identify proteins recruited to the cccPML-NBs. Based on our recent work (Hofmann, Schreiner and EU Patent), in AIM3 we propose that specific interference with the SUMOylation of viral substrates and viral dependency on functional PML-NBs promoting infection will provide an efficient target for novel treatment options. Therefore, we aim to elucidate the efficacy of specific anti-SUMO/PML-NB drugs in our HBV infection models, in authentic in vitro 3D-organoids (with Dao Thi) and in in vivo HBV mouse models (with Protzer/Kosinska, Protzer/Wettengel, Wohlleber). Further, we will work on the generation of HBV core/HBx/HBV pol SUMOylation specific inhibitors and PROTAC ubiquitinylation assays to evaluate these antiviral intervention options for their therapeutic potential.