Sprecher SFB 1021

Intracellular organization of Ebola virus RNA synthesis; formation, maturation and transport of viral RNP

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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:

  • 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. J Virol 94.
  • 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.
  • 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. Proc Natl Acad Sci U S A 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. J Infect Dis 218: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:S160-6.
  • 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 Pathog 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. Proc Natl Acad Sci U S A 110:14402-7.
  • 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. Proc Natl Acad Sci U S A 109:4275-80.

Adaptation of Marburg virus to rodents as a model to study viral pathogenesis: Response of the mononuclear phagocyte system

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Research area: Molecular Virology

Marburg virus (MARV) causes lethal fever in humans and non-lethal infections in rodents. Sequential passaging of MARV in guinea pigs resulted in a guinea pig-adapted strain that contains 4 non-synonymous mutations and causes lethal disease. Cells of the mononuclear phagocyte system (MPS) were found to be the primary MARV targets in both lethal and non-lethal infection. The Becker group discovered that a single mutation in the viral matrix protein VP40 (D184N) improved the growth of recombinant MARV (rMARVVP40(D184N)) specifically in guinea pig cells. This phenotype correlated with a decreased ability of VP40D184N to suppress viral RNA synthesis in these cells. In primary MPS cells derived from a newly established ex vivo model, rMARVVP40(D184N) had a clear growth advantage over wild-type MARV. This correlated with the observation that rMARVVP40(D184N), in contrast to the non-lethal wild type virus, no longer induced cell death in the infected macrophages or in non-infected bystander cells. Other mutations in the guinea pig-adapted MARV were found to be located in the polymerase (L) coding sequence. One of the mutations located in the polymerase active site was shown to dramatically increase the viral transcription/replication activity.

Project-related publications of the investigator:

  • Dietzel E, Schudt G, Krähling V, Matrosovich M, Becker S. 2016. Functional characterization of adaptive mutations during the West African Ebola virus outbreak. J. Virol. 91:e01913-16.
  • Koehler A, Kolesnikova L, Becker S. 2016. An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus. J Gen Virol. 97(10):2494-2500.
  • Koehler A, Kolesnikova L, Welzel U, Schudt G, Herwig A, Becker S. 2016. A Single Amino Acid Change in the Marburg Virus Matrix Protein VP40 Provides a Replicative Advantage in a Species-Specific Manner. J Virol 90:1444-1454.
  • Wolff S, Groseth A, Meyer B, Jackson D, Strecker T, Kaufmann A, Becker S. 2016. The New World arenavirus Tacaribe virus induces caspase-dependent apoptosis in infected cells. J Gen Virol 97:855-866.
  • Dietzel E, Kolesnikova L, Sawatsky B, Heiner A, Weis M, Kobinger GP, Becker S, von Messling V, Maisner A. 2015. Nipah virus matrix protein influences fusogenicity and is essential for particle infectivity and stability. J Virol. 90(5):2514-22.
  • Schudt, G., O. Dolnik, L. Kolesnikova, N. Biedenkopf, A. Herwig and S. Becker. 2015. Transport of Ebolavirus Nucleocapsids Is Dependent on Actin Polymerization: Live-Cell Imaging Analysis of Ebolsvirus-Infected Cells. J. Infect Dis doi: 10.1093/infdis/jiv083
  • Kolesnikova, L., E. Mittler, G. Schudt, H. Shams-Eldin, and S. Becker. 2012. Phosphorylation of Marburg virus matrix protein VP40 triggers assembly of nucleocapsids with the viral envelope at the plasma membrane. Cell Microbiol 14:182-97
  • Mateo, M., C. Carbonnelle, O. Reynard, L. Kolesnikova, K. Nemirov, A. Page, V. A. Volchkova, and V. E. Volchkov. 2011. VP24 is a molecular determinant of Ebola virus virulence in guinea pigs. J Infect Dis 204 Suppl 3:S1011-20.
  • Welsch, S., L. Kolesnikova, V. Krahling, J. D. Riches, S. Becker, and J. A. Briggs. 2010. Electron tomography reveals the steps in filovirus budding. PLoS Pathog 6:e1000875.
  • Krahling, V., O. Dolnik, L. Kolesnikova, J. Schmidt-Chanasit, I. Jordan, V. Sandig, S. Gunther, and S. Becker. 2010. Establishment of fruit bat cells (Rousettus aegyptiacus) as a model system for the investigation of filoviral infection. PLoS Negl Trop Dis 4:e802.
  • Dolnik, O., L. Kolesnikova, and S. Becker. 2008. Filoviruses: Interactions with the host cell. Cell Mol Life Sci 65:756-76.
  • Kolesnikova, L., A. B. Bohil, R. E. Cheney, and S. Becker. 2007. Budding of Marburgvirus is associated with filopodia. Cell Microbiol 9:939-51.
  • Kolesnikova, L., B. Berghofer, S. Bamberg, and S. Becker. 2004. Multivesicular bodies as a platform for formation of the Marburg virus envelope. J Virol 78:12277-87.
  • Kolesnikova, L., S. Bamberg, B. Berghofer, and S. Becker. 2004. The matrix protein of Marburg virus is transported to the plasma membrane along cellular membranes: exploiting the retrograde late endosomal pathway. J Virol 78:2382-93.
  • Ryabchikova, E. I., L.  Kolesnikova, and S. V. Luchko. 1999. An analysis of features of pathogenesis in two animal models of Ebola virus infection. J Infect Dis 179 Suppl 1:S199-202.
  • Mühlberger, E., B. Lotfering, H.-D. Klenk, and S. Becker. 1998. Three of the four nucleocapsid proteins of Marburg virus, NP, VP35, and L, are sufficient to mediate replication and transcription of Marburg virus-specific monocistronic minigenomes. J Virol 72:8756-64.
  • Ryabchikova, E., L. Kolesnikova, M. Smolina, V. Tkachev, L. Pereboeva, S. Baranova, A. Grazhdantseva, and Y. Rassadkin. 1996. Ebola virus infection in guinea pigs: presumable role of granulomatous inflammation in pathogenesis. Arch Virol 141:909-21.
  • Ryabchikova, E., L. Strelets, L. Kolesnikova, O. Pyankov, and A. Sergeev. 1996. Respiratory Marburg virus infection in guinea pigs. Arch Virol 141:2177-90.