• 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:

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


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
E-Mail:
becker(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:
  • Takamatsu Y, Krähling V, Kolesnikova L, Halwe S, Lier C, Baumeister S, Noda T, Biedenkopf NBecker S. Serine-arginine protein kinase 1 regulates Ebola virus transcription. mBio 2020 accepted. MSID mBio02565-19R1
  • Bach S, Biedenkopf N, Grünweller A, Becker SHartmann RK, Hexamer phasing governs transcription initiation in the 3´ leader of Ebola virus. RNA 2020 accepted, MS ID RNA/2019/073718
  • Kruse T, Biedenkopf N, Hertz EPT, Dietzel E, Stalmann G, López-Méndez B, Davey NE, Nilsson J, Becker S. The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30. Molecular Cell. 2018 Jan 4;69(1):136-145.e6.
  • Lier C, Becker SBiedenkopf N. Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies. Virology. 2017 Dec;512:39-47.
  • Biedenkopf N, Lange-Grünweller K, Schulte FW, Weißer A, Müller C, Becker D, Becker SHartmann RK, Grünweller A. The natural compound silvestrol is a potent inhibitor of Ebola virus replication. Antiviral Research. 2017 Jan;137:76-81. doi: 10.1016/j.antiviral.2016.11.011. Epub 2016 Nov 15.
  • Schlereth J, Grünweller A, Biedenkopf N, Becker SHartmann RK. RNA binding specificity of Ebola virus transcription factor VP30. RNA Biology. 2016 Sep;13(9):783-98. doi: 10.1080/15476286.2016.1194160. Epub 2016 Jun 17.
  • Biedenkopf N, Schlereth J, Grünweller A, Becker SHartmann RK. RNA Binding of Ebola Virus VP30 Is Essential for Activating Viral Transcription. Journal of Virology. 2016 Jul 27;90(16):7481-7496. doi: 10.1128/JVI.00271-16. Print 2016 Aug 15.
  • Biedenkopf N, Lier C, Becker S. Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle. Journal of Virology. 2016 Apr 29;90(10):4914-4925. doi: 10.1128/JVI.03257-15. Print 2016 May 15
  • Biedenkopf N, Hartlieb B, Hoenen T, Becker S. Phosphorylation of Ebola virus VP30 influences the composition of the viral nucleocapsid complex: Impact on viral transcription and replication. Journal of Biological Chemistry, March 2013, doi 10.1074/jbc.M113.461285
  • Martinez MJ, Biedenkopf N, Volchkova V, Hartlieb B, Alazard-Dany N, Reinard O, Becker S, Volchkov V. Role of Ebola virus VP30 in Transcription Reinitiation. Journal of Virology. 2008 Dec;82(24):12569-73. doi: 10.1128/JVI.01395-08. Epub 2008 Oct 1.
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.
med.uni-giessen(dot)de


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, Roeb E, Michel AM, Niepmann M. Hepatitis C Virus Downregulates Core Subunits of Oxidative Phosphorylation, Reminiscent of the Warburg Effect in Cancer Cells. Cells. 2019 Nov 8;8(11). pii: E1410. doi: 10.3390/cells8111410. Review (includes also previously unpublished original data).
  • Hu P, Wilhelm J, Gerresheim GK, Shalamova LA, Niepmann M. Lnc-ITM2C-1 and GPR55 Are Proviral Host Factors for Hepatitis C Virus. Viruses. 2019 Jun 13;11(6). pii: 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. Cellular Gene Expression during Hepatitis C Virus Replication as Revealed by Ribosome Profiling. Int J Mol Sci. 2019 Mar 15;20(6). pii: E1321.
  • Niepmann M, Shalamova LA, Gerresheim GK, Rossbach O. Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication. Front Microbiol. 2018 Mar 12;9:395. doi: 10.3389/fmicb.2018.00395. eCollection 2018. Review.
  • Jost I, Shalamova LA, Gerresheim GK, Niepmann M, Bindereif A, Rossbach O. Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges. RNA Biol. 2018;15(8):1032-1039.
  • Nieder-Röhrmann A, Dünnes N, Gerresheim GK, Shalamova LA, Herchenröther A, Niepmann M. 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. 2017 Feb;98(2):212-224.
  • Gerresheim GK, Dünnes N, Nieder-Röhrmann A, Shalamova LA, Fricke M, Hofacker I, Höner Zu Siederdissen C, Marz M, Niepmann M. 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. 2017 Feb;74(4):747-760.
  • Fricke M, Dünnes N, Zayas M, Bartenschlager R, Niepmann M, Marz M. Conserved RNA secondary structures and long-range interactions in hepatitis C viruses. RNA. 2015 Jul;21(7):1219-32. doi: 10.1261/rna.049338.114.
  • Henke JI, Goergen D, Zheng J, Song Y, Schüttler CG, Fehr C, Jünemann C, Niepmann M. microRNA-122 stimulates translation of hepatitis C virus RNA. EMBO J. 2008 Dec 17;27(24):3300-10.
  • Song Y, Friebe P, Tzima E, Jünemann C, Bartenschlager R, Niepmann M. The hepatitis C virus RNA 3'-untranslated region strongly enhances translation directed by the internal ribosome entry site. J Virol. 2006 Dec;80(23):11579-88.