• Projects

Project area A:

Structure and Metabolism of viral RNA

Principal investigator:

Prof. Dr. John Ziebuhr

Institut für Medizinische Virologie
Justus-Liebig-Universität Gießen
Schubertstraße 81
35392 Gießen

Phone: 0641-99 41200
E-Mail: john.ziebuhr(at)viro.med.uni-giessen(dot)de

Research area: Molecular Virology
The order Nidovirales (families Coronaviridae, Mesoniviridae, Arteriviridae and Roniviridae) comprises viruses with extremely large RNA genomes and includes important animal and human pathogens, such as SARS and MERS coronavirus. The nidovirus replication/transcription complex (RTC) consists of an exceptionally large number of viral enzymes. This also includes a 3'-5' exoribonuclease (ExoN) presumed to be involved in proofreading mechanisms during viral replication. In the first funding period, the Ziebuhr group characterized the biochemical properties of this enzyme for representative viruses of the subfamilies Corona- and Torovirinae and has extended these studies to other nonstructural proteins (nsp7, 8, 9, 10, 12, 13) that are thought to form the coronavirus/nidovirus "core replicase". Furthermore, essential cis-active RNA elements conserved across the genus Alphacoronavirus were identified and a detailed characterization of the replication/transcription complex of the newly discovered Mesoniviridae was initiated. In this context, the Ziebuhr group characterized the mesonivirus main protease and characterized the proteolytic processing of the mesonivirus "core" replicase. Together, these studies provide the basis for comparative studies of common and distinct mechanisms involved in the replication of nidoviruses with large- (30 kb) and medium-size (20 kb) RNA genomes using biochemical and reverse-genetics approaches.
Project-related publications of the investigator:
  • Kanitz M, Blanck S, Heine A, Gulyaeva AA, Gorbalenya AE, Ziebuhr J*, Diederich WE*. Structural basis for catalysis and substrate specificity of a 3C-like cysteine protease from a mosquito mesonivirus. Virology 2019; 533:21-33. *corresponding authors
  • Tvarogová J, Madhugiri R, Bylapudi G, Ferguson LJ, Karl N, Ziebuhr J. Identification and characterization of a human coronavirus 229E nonstructural protein 8-associated RNA 3'-terminal adenylyltransferase activity. J Virol 2019; 93:e00291-19.
  • Durzynska I, Sauerwald M, Karl N, Madhugiri R, Ziebuhr J. Characterization of a bafinivirus exoribonuclease activity. J Gen Virol 2018; 99:1253-1260.
  • Madhugiri R, Karl N, Petersen D, Lamkiewicz K, Fricke M, Wend U, Scheuer R, Marz M, Ziebuhr J. Structural and functional conservation of cis-acting RNA elements in coronavirus 5'-terminal genome regions. Virology 2018; 517:44-55.
  • Müller C, Hardt M, Schwudke D, Neuman BW, Pleschka S, Ziebuhr J. Inhibition of Cytosolic phospholipase A2alpha impairs an early step of coronavirus replication in cell culture. J Virol 2018; 92:01463-17.
  • Kindler E, Gil-Cruz C, Spanier J, Li Y, Wilhelm J, Rabouw HH, Zust R, Hwang M, V'Kovski P, Stalder H, Marti S, Habjan M, Cervantes-Barragan L, Elliot R, Karl N, Gaughan C, van Kuppeveld FJ, Silverman RH, Keller M, Ludewig B, Bergmann CC, Ziebuhr J, Weiss SR, Kalinke U, Thiel V. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication. PLoS Pathog 2017; 13:e1006195.
  • Poppe M, Wittig S, Jurida L, Bartkuhn M, Wilhelm J, Müller H, Beuerlein K, Karl N, Bhuju S, Ziebuhr J, Schmitz ML, Kracht M. The NF-kappaB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells. PLoS Pathog 2017; 13:e1006286.
  • Snijder EJ, Decroly E, Ziebuhr J. The nonstructural proteins directing coronavirus RNA synthesis and processing. Adv Virus Res 2016; 96:59-126.
  • Madhugiri R, Fricke M, Marz M, Ziebuhr J. Coronavirus cis-acting RNA elements. Adv Virus Res 2016; 96:127-163.
  • Minskaia E, Hertzig T, Gorbalenya AE, Campanacci V, Cambillau C, Canard B, Ziebuhr J. Discovery of an RNA virus 3'-5' exoribonuclease that is critically involved in coronavirus RNA synthesis. Proc Natl Acad Sci USA 2006; 103:5108-5113.
Principal investigator:

Dr. Nadine Biedenkopf

Institut für Virologie
Philipps-Universität Marburg
Hans-Meerwein-Str. 2
35043 Marburg

Phone: 06421-28 25307

Principal investigator:

Prof. Dr. Roland Hartmann

Institut für Pharmazeutische Chemie
Philipps-Universität Marburg
Marbacher Weg 6
35037 Marburg

Phone: 06421-28 25827
E-Mail: roland.hartmann(at)staff.uni-marburg(dot)de

Research areas: Molecular Virology, RNA Biochemistry

This project investigates factors and molecular mechanisms involved in Ebola virus (EBOV) transcription, focusing on the EBOV-specific transcription factor VP30, a hexameric phosphoprotein that contains a zinc-finger domain with RNA-binding activity. Viral transcription factor activity of VP30 was previously shown to be largely controlled by the phosphorylation state of this protein. In the first funding period, the Becker and Hartmann groups investigated the RNA-binding and transcriptional regulation activities mediated by VP30. They found that all functions of VP30 involved in RNA binding, such as VP30 phosphorylation, homooligomerization, and the integrity of a zinc finger motif, simultaneously affect the protein’s ability to activate transcription. Interaction between VP30 and the polymerase cofactor VP35 was shown to depend on RNA binding and on the dsRNA binding activity of VP35. VP30 preferentially interacted with single-stranded RNA substrates of mixed base composition; a stem-loop structure, particularly at the 3'-end, further stabilized this binding. RNA binding of VP30 was impaired in the presence of a 5´-Cap(0) structure, and phosphorylation of VP30 inhibited RNA binding. Interestingly, the full transcriptional support activity of VP30 was dependent on a dynamic sequence of phosphorylation/dephosphorylation of N-terminal serine residues, suggesting that both phosphorylated and nonphosphorylated VP30 are essential for different steps of transcription.

Project-related publications of the investigators:
  • Bach S, Demper JC, Grünweller A, Becker S, Biedenkopf N, Hartmann RK. 2020c. Regulation of VP30-dependent transcription by RNA sequence and structure in the genomic Ebola virus promoter. J Virol accepted as JVI02215-20.
  • Bach S, Demper JC, Biedenkopf N, Becker S, Hartmann RK. 2020b. RNA secondary structure at the transcription start site influences EBOV transcription initiation and replication in a length- and stability-dependent manner. RNA Biol:1-14.
  • Bach S, Biedenkopf N, Grünweller A, Becker S, Hartmann RK. 2020a. Hexamer phasing governs transcription initiation in the 3'-leader of Ebola virus. RNA 26:439-453.
  • Takamatsu Y, Krähling V, Kolesnikova L, Halwe S, Lier C, Baumeister S, Noda T, Biedenkopf N, Becker S. 2020. Serine-Arginine Protein Kinase 1 Regulates Ebola Virus Transcription. mBio 11.
  • Kruse T, Biedenkopf N, Hertz EPT, Dietzel E, Stalmann G, López-Méndez B, Davey NE, Nilsson J, Becker S. 2018. The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30. Mol Cell 69:136-145.e6.
  • Biedenkopf N, Hoenen T. 2017. Modeling the Ebolavirus Life Cycle with Transcription and Replication-Competent Virus like Particle Assays. Methods Mol Biol 1628:119-131.
  • Lier C, Becker S, Biedenkopf N. 2017. Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies. Virology 512:39-47.
  • Biedenkopf N, Lange-Grünweller K, Schulte FW, Weißer A, Müller C, Becker D, Becker S, Hartmann RK, Grünweller A. 2017. The natural compound silvestrol is a potent inhibitor of Ebola virus replication. Antiviral Res 137:76-81.
  • Biedenkopf N, Schlereth J, Grünweller A, Becker S, Hartmann RK. 2016. RNA Binding of Ebola Virus VP30 Is Essential for Activating Viral Transcription. J Virol 90:7481-7496.
  • Schlereth J, Grünweller A, Biedenkopf N, Becker S, Hartmann RK. 2016. RNA binding specificity of Ebola virus transcription factor VP30. RNA Biol 13:783-98.
Principal investigator:

Prof. Dr. Michael Niepmann

Biochemisches Institut
Justus-Liebig-Universität Gießen
Friedrichstraße 24
35392 Gießen

Phone: 0641-99 47471
E-Mail: michael.niepmann(at)biochemie.

Research area: Molecular Virology

To analyze hepatitis C virus minus- and plus-strand synthesis initiation, the Niepmann group developed a split replication system in which the functions of both genome ends can be studied separately. This system differs from previously established HCV replicon systems that, in all cases, required the presence of both genome ends and used genome amplification to monitor viral RNA synthesis. The split replication system provides new and unprecedented insights into HCV replication mechanisms and is of great scientific interest because it is the first system that allows specific cis-acting signals to be linked to specific functions in minus- and plus-strand RNA synthesis, respectively, while uncoupling these functions from overlapping functions of the involved sequences in other steps of the viral life cycle.

Project-related publications of the investigator:
  • Gerresheim GK*, Hess CS, Shalamova LA*, Fricke M, Marz M, Andreev DE, Shatsky IN, and Niepmann M. 2020. Ribosome pausing at inefficient codons at the end of the replicase coding region is important for Hepatitis C Virus genome replication. Int J Mol Sci; 21:E6955.
  • Niepmann M, Gerresheim GK*. Hepatitis C Virus Translation Regulation. 2020. Int J Mol Sci; 21:2328. Review.
  • Gerresheim GK*, Roeb E, Michel AM, Niepmann M. 2019. Hepatitis C Virus Downregulates Core Subunits of Oxidative Phosphorylation, Reminiscent of the Warburg Effect in Cancer Cells. Cells; 8. pii: E1410. Review (includes also previously unpublished original data).
  • Hu P, Wilhelm J, Gerresheim GK*, Shalamova LA, Niepmann M. 2019. Lnc-ITM2C-1 and GPR55 Are Proviral Host Factors for Hepatitis C Virus. Viruses; 11:E549.
  • Gerresheim GK*, Bathke J, Michel AM, Andreev DE, Shalamova LA*, Rossbach O, Hu P, Glebe D, Fricke M, Marz M, Goesmann A, Kiniry SJ, Baranov PV, Shatsky IN, Niepmann M. 2019. Cellular Gene Expression during Hepatitis C Virus Replication as Revealed by Ribosome Profiling. Int J Mol Sci; 20:E1321.
  • Fricke M, Gerst R, Ibrahim B, Niepmann M, Marz M. 2019. Global importance of RNA secondary structures in protein-coding sequences. Bioinformatics; 35:579.
  • Niepmann M, Shalamova LA*, Gerresheim GK*, Rossbach O. 2018. Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication. Front Microbiol; 9:395. Review.
  • Jost I, Shalamova LA*, Gerresheim GK*, Niepmann M, Bindereif A, Rossbach O. 2018. Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges. RNA Biol; 15:1032.
  • Nieder-Röhrmann A, Dünnes N, Gerresheim GK, Shalamova LA, Herchenröther A, Niepmann M. 2017. Cooperative enhancement of translation by two adjacent microRNA-122/Argonaute 2 complexes binding to the 5' untranslated region of hepatitis C virus RNA. J Gen Virol; 98:212.
  • Gerresheim GK, Dünnes N, Nieder-Röhrmann A, Shalamova LA, Fricke M, Hofacker I, Höner Zu Siederdissen C, Marz M, Niepmann M. 2017. microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure. Cell Mol Life Sci; 74:747.
Principal investigator:

Prof. Dr. Stephan Becker

Sprecher SFB 1021

Institut für Virologie
Philipps-Universität Marburg
Hans-Meerwein-Str. 2
35043 Marburg

Phone: 06421-28 66253

Research area: Molecular Virology

Replication of viral genomic RNA results in the formation of progeny genomes that need to be packaged and transported to the sites of viral budding. Replication of negative-strand Ebola virus genomes takes place in inclusion bodies located close to the nucleus. During synthesis, genomes are encapsidated to form ribonucleoprotein (RNP) complexes which mature to transport-competent nucleocapsids (NCs) inside the inclusions. Although the virus-induced inclusions play an important role in the synthesis of filovirus RNA, their organization is not well understood. In the proposed project we will investigate the spatial and temporal organization of filoviral RNA synthesis, its packaging into RNPs, the molecular basis for RNP maturation to become transport-competent NCs, and which cellular and viral factors are involved in the recruitment of the actin-polymerizing machinery that drives the transport of NCs. Results gained with surrogate systems under BSL-2 conditions will be validated using recombinant filoviruses and CRISPR/Cas9 knock out cell lines under BSL-4 conditions. We will use proteomics to identify cellular proteins associated with encapsidated genomes, quantitative live cell imaging to understand the transport dynamics, super resolution microscopy (dSTORM), and correlative light and electron microscopy (CLEM) to visualize actin and actin-binding proteins at the NCs.

Project-related publications of the investigator:
  • Grikscheit K, Dolnik O, Takamatsu Y, Pereira AR, Becker S. 2020. Ebola Virus Nucleocapsid-Like Structures Utilize Arp2/3 Signaling for Intracellular Long-Distance Transport. Cells 9:1728
  • Takamatsu Y, Kolesnikova L, Schauflinger M, Noda T, Becker S. 2020. The Integrity of the YxxL Motif of Ebola Virus VP24 Is Important for the Transport of Nucleocapsid-Like Structures and for the Regulation of Viral RNA Synthesis. Journal of Virology 94: e02170-19.
  • Takamatsu Y, Dolnik O, Noda T, Becker S. 2019. A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures. Virol J 16:159.
  • Takamatsu Y, Kolesnikova L, Becker S. 2018. Ebola virus proteins NP, VP35 and VP24 are essential and sufficient to mediate nucleocapsid transport. PNAS 115: 1075-1080
  • Mittler E, Schudt G, Halwe S, Rohde C, Becker S. 2018. A Fluorescently Labeled Marburg Virus Glycoprotein as a New Tool to Study Viral Transport and Assembly. Journal Infect Dis 2018: S318-S326
  • Wan W, Kolesnikova L, Clarke M, Koehler A, Noda T, Becker S, Briggs JAG. 2017. Structure and assembly of the Ebola virus nucleocapsid. Nature 551:394-397.
  • Schudt G, Dolnik O, Kolesnikova L, Biedenkopf N, Herwig A, Becker S. 2015. Transport of Ebolavirus Nucleocapsids Is Dependent on Actin Polymerization: Live-Cell Imaging Analysis of Ebolavirus-Infected Cells. J Infect Dis 212 Suppl 2:S160-166.
  • Dolnik O, Kolesnikova L, Welsch S, Strecker T, Schudt G, Becker S. 2014. Interaction with Tsg101 is necessary for the efficient transport and release of nucleocapsids in Marburg virus-infected cells. PLoS pathogens 10:e1004463.
  • Schudt G, Kolesnikova L, Dolnik O, Sodeik B, Becker S. 2013. Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances. PNAS 110:14402-14407.
  • Bharat TA, Noda T, Riches JD, Kraehling V, Kolesnikova L, Becker S, Kawaoka Y, Briggs JA. 2012. Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography. PNAS 109:4275-4280.