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