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Carola Busch, Sophia Reindl, Tobias Holm, Dominik Vogel, Maria Rosenthal, Nadja Gogrefe
We want to understand the molecular and structural mechanisms how segmented negative strand RNA viruses, such as Lassa Virus or Rift Valley Fever Virus, use their own proteins and interact with cellular proteins in order to replicate and transcribe their RNA genome.
Viruses from the order of segmented negative stand RNA viruses produce only a handful of own proteins, some of them get by with only four gene products. Nonetheless several of the most deadly and newly emerging pathogens like the Lassa Virus and the Crimean-Congo hemorrhagic fever Virus belong to this group. Another virus, the Rift-Valley-Fever (RVF) Virus, has been known for approx. 100 years, but large RVF outbreaks in humans have been reported more and more often during the last years. Thus in addition to the newly emerged viruses, some of the long-known viruses gained a more important role for public health. How these small viruses are able to use their 4-6 proteins in a way that allows them to have such a dramatic effect on their host is fascinating and not yet understood.
Two proteins are mainly in the focus of our research: The L protein and the N protein that together with the RNA genome of the virus form the Ribonucleoparticle, which is sufficient for replication and transcription of the genome. We solved the structure of the N protein from Lassa Virus at atomic resolution and obtained the first glimpse at an important part of the replication complex.
Now we concentrate on the large multidomain L protein and its interaction with N. We produce the proteins in a suitable expression system, e.g. bacteria, insect cells or mammalian cells. We then use an interdisciplinary approach combining structural biology (protein crystallography and Small angle X-ray scattering) with functional studies from in vitro enzyme activity assays and cell-based replication and transcription experiments to understand catalytic mechanisms and enzyme dynamics.
Besides the central polymerase domain, the L protein also contains an endonuclease in its N-terminus. Hantaviruses, another group of important pathogens from the familiy of Bunyaviridae, contain an endonuclease, which is too active to be recombinantly produced: it degrades all RNA making it toxic for the expressing cell. We studied the endonuclease in detail by introducing a set of mutations, which result in an attenuated enyzme, allowing for expression in E. coli. By determination of the crystal structure we could propose a role of all mutated amino acids and gain new insights into an enzyme, which is essential for the viral life cycle and therefore an attractive drug target.
Structural insights into reptarenavirus cap-snatching machinery.
Rosenthal M, Gogrefe N, Vogel D, Reguera J, Rauschenberger B, Cusack S, Günther S, Reindl S
PLoS Pathog. 2017 May 15;13(5):e1006400. doi: 10.1371/journal.ppat.1006400.
Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.
Fernández-García Y, Reguera J, Busch C, Witte G, Sánchez-Ramos O, Betzel C, Cusack S, Günther S, Reindl S.
PLoS Pathog. 2016 Jun 14;12(6):e1005635.
Comparative Structural and Functional Analysis of Bunyavirus and Arenavirus Cap-Snatching Endonucleases.
Reguera J, Gerlach P, Rosenthal M, Gaudon S, Coscia F, Günther S, Cusack S.
PLoS Pathog. 2016 Jun 15;12(6):e1005636.
Role of the C Terminus of Lassa Virus L Protein in Viral mRNA Synthesis.
Lehmann M, Pahlmann M, Jerome H, Busch C, Lelke M, Günther S.
J Virol. 2014 Aug;88(15):8713-7. doi: 10.1128/JVI.00652-14.
The N terminus of Andes virus L protein suppresses mRNA and protein expression in mammalian cells.
Heinemann P, Schmidt-Chanasit J, Günther S.
J Virol. 2013 Jun;87(12):6975-85. doi: 10.1128/JVI.00043-13.
Structure of the Lassa virus nucleoprotein revealed by X-ray crystallography, small-angle X-ray scattering, and electron microscopy.
Brunotte L, Kerber R, Shang W, Hauer F, Hass M, Gabriel M, Lelke M, Busch C, Stark H, Svergun DI, Betzel C, Perbandt M, Günther S.
J Biol Chem. 2011 Nov 4;286(44):38748-56. doi: 10.1074/jbc.M111.278838.
Domain structure of Lassa virus L protein.
Brunotte L, Lelke M, Hass M, Kleinsteuber K, Becker-Ziaja B, Günther S.
J Virol. 2011 Jan;85(1):324-33. doi: 10.1128/JVI.00721-10.
The N-terminal domain of the arenavirus L protein is an RNA endonuclease essential in mRNA transcription.
Morin B, Coutard B, Lelke M, Ferron F, Kerber R, Jamal S, Frangeul A, Baronti C, Charrel R, de Lamballerie X, Vonrhein C, Lescar J, Bricogne G, Günther S, Canard B.
PLoS Pathog. 2010 Sep 16;6(9):e1001038. doi: 10.1371/journal.ppat.1001038.