TP24: Functional analysis of molecular perturbations induced by Hepatitis B and D virus infection

Using advanced proteomics analysis, we found that host protein turnover (protein stability and degradation) is critical to regulate the interferon-mediated cellular antiviral defence. Moreover, in HBV infected cells we could identify ~100 proteins that show differential turnover rates after infection and evaluated 30 candidates for their ability to regulate HBV DNA and cccDNA abundance. Our data revealed a surprising relationship between proteins involved in energy metabolism (COX6C and NDUFB9) and the selective accumulation of cccDNA. We will further characterize this relationship and extend our studies to clinically important HBV/HDV co-infection models, which we will functionally evaluate with the aim to identify yet undescribed molecular mechanisms that determine virus proliferation and persistence.

Prof. Dr. Dr. Andreas Pichlmair

Hubel P, Urban C, Bergant V, Schneider WM, Knauer B, Stukalov A, Scaturro P, Mann A, Brunotte L, Hoffmann HH, Schoggins JW, Schwemmle M, Mann M, Rice CM, Pichlmair A. (2019) A protein-interaction network of interferon-stimulated genes extends the innate immune system landscape. Nat Immunol. 20, 493-502. doi:10.1038/s41590-019-0323-3.

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.

Urban C, Welsch H, Heine K, Wust S, Haas DA, Dachert C, Pandey A, Pichlmair A, Binder M. (2020) Persistent Innate Immune Stimulation Results in IRF3-Mediated but Caspase-Independent Cytostasis. Viruses. 12. doi:10.3390/v12060635.

Burkart SS, Schweinoch D, Frankish J, Sparn C, Wust S, Urban C, Merlo M, Magalhaes VG, Piras A, Pichlmair A, Willemsen J, Kaderali L, Binder M. (2023) High-resolution kinetic characterization of the RIG-I-signaling pathway and the antiviral response. Life Sci Alliance. 6. doi:10.26508/lsa.202302059.

Laudenbach BT, Krey K, Emslander Q, Andersen LL, Reim A, Scaturro P, Mundigl S, Dachert C, Manske K, Moser M, Ludwig J, Wohlleber D, Kroger A, Binder M, Pichlmair A. (2021) NUDT2 initiates viral RNA degradation by removal of 5'-phosphates. Nat Commun. 12, 6918. doi:10.1038/s41467-021-27239-y.

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, Wolfel 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, 246-252. doi:10.1038/s41586-021-03493-4.

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, Strotges-Achatz A, Haselmann V, Neumaier M, Erber J, Priller A, Yazici S, Roggendorf H, Odendahl M, Tonn T, Dick A, Witter K, Mijocevic 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. 38, 110214. doi:10.1016/j.celrep.2021.110214.

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.

Lampl S, Janas MK, Donakonda S, Brugger M, Lohr K, Schneider A, Manske K, Sperl LE, Klager S, Kuster B, Wettmarshausen J, Muller C, Laschinger M, Hartmann D, Huser N, Perocchi F, Schmitt-Kopplin P, Hagn F, Zender L, Hornung V, Borner C, Pichlmair A, Kashkar H, Klingenspor M, Prinz M, Schreiner S, Conrad M, Jost PJ, Zischka H, Steiger K, Kronke M, Zehn D, Protzer U, Heikenwalder M, Knolle PA, Wohlleber D. (2020) Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signaling in virus-infected hepatocytes. J Hepatol. 73, 1347-1359. doi:10.1016/j.jhep.2020.06.026.

Scaturro P, Stukalov A, Haas DA, Cortese M, Draganova K, Plaszczyca A, Bartenschlager R, Gotz M, Pichlmair A. (2018) An orthogonal proteomic survey uncovers novel Zika virus host factors. Nature. 561, 253-257. doi:10.1038/s41586-018-0484-5.

Project in detail +

Given the highly promising results of the second funding period and related to the identification of novel HBV host cell dependency factors, we will characterize in-depth the molecular mode-of-action of these factors with the aim to gain mechanistic insights. In parallel, we will expand our proteomics analysis to HDV infections and HBV/HDV co-infections since interactions between these viruses and the host cell are insufficiently understood, but may provide insights into the regulation between replication and persistence between these viruses and potentially identify novel targets for antiviral therapy to regulate these processes. Using the established proteomics approach, we aim to identify dynamic changes of the host proteome that enable unique insights into regulatory mechanisms operating in HDV and HBV/HDV infected primary cells. The goal is to identify proteins and pathways that promote or inhibit HBV, HDV or HBV/HDV replication or may explain the disease-promoting activity of HDV in HBV/HDV co-infections. Using loss of function (CRISPR/Cas9, siRNA) and drug perturbation experiments, we will functionally and mechanistically dissect underlying mechanisms as well as the consequences triggered by virus-induced perturbations. Taking advantage of the unique expertise within TRR179, this project will benefit from expertise in HBV and HDV cell biology (Bartenschlager, Knolle, Protzer/Wettengel, Urban/Nkongolo) as well as infection-associated responses in immune cells (Bengsch, Cerwenka, Hofmann, Thimme, Wohlleber, Neumann-Haefelin/Luxenburger). Moreover, the analysis of protein expression patterns will benefit from integrating data gathered by the single cell transcriptome and histological analyses that are planned to be generated within the Z02 and Z03 as part of their attempt to establish a multiomics cell atlas of the hepatitis virus infected liver. Importantly, we expect to identify yet unknown regulatory processes that may contribute to the spread and persistence of HBV and HDV as well as possible liver-disease promoting processes that are operative in hepatitis virus infections.