rhdr

Efe Can Yildirim, Kristina Meier, Dominik Vogel, Silke Olschewski, Maria Rosenthal, Nadja Gogrefe, Carola Busch

Overview

Arenavirus particle – the L protein is structurally mainly uncharacterized

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.

 

 

 

 

Applications of motivated students are always welcome! If you are interested in biochemistry and structural biology in context of virus research, please apply via email to rosenthal@bnitm.de.

Research Projects

Protein crystal

Replication machinery of segmented negative strand RNA viruses 

Viruses from the order of bunyavirales with a segmented negative strand RNA genome 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.

Symmetric trimer of the Lassa Nucleoprotein crystal structure superimposed with the class average view from negative stain EM.

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 endoribonuclease in its N-terminus. Hantaviruses, another group of important pathogens from the order of bunyavirales, 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. 

The endonuclease activity in the N-terminus of the hantaviral L protein. A: Expression level of full L protein variants and their mRNA in mammalian cells. B: Endonuclease activity of recombinantly produced isolated endonuclease domain variants of the L protein. Radioactively labelled RNA is separated by denatured PAGE. C: Crystal structure of the hantaviral endonuclease domain. All residues mutated in A are shown and grouped by their proposed role.

In the recent years we solved further structures of fragments of the large L protein: the N- and C-terminal domains of the California Academy of Sciences virus (reptarenavirus) L protein. The N-terminal domain of the L protein contains an endoribonuclease, which is structurally homologous to already published arenavirus endonucleases. The C-terminus of the L protein contains a putative cap-binding site very similar to influenza virus cap-binding protein PB2. Both of these functions, cap-binding and endonuclease, are required for the cap-snatching mechanism, by which segmented negative strand RNA viruses presumably initiate transcription. In our structure however, the residues potentially involved in cap-binding did not show the expected conformation and also functional studies could not proof a cap-binding activity. Thus it remains still unclear whether the required cap-binding function is present in other L proteins of viruses from the bunyavirales order.

Left: The structure of the N-terminus of reptarenavirus CASV L protein (A) is similar to other endonucleases of mammarenavirus L proteins (C). The active site comformation is almost identical (B).

Right: The structure of the C-terminus of CASV L protein contains two separate domains (A) and is very similar to parts of influenza virus PB2, even concerning the domain topology (B). This is exemplarily shown for domain 2 of CASV L protein C-terminus and PB2 cap-binding domain (C).

All Publications

Department of Virology

Biochemical characterization of the Lassa virus L protein.
Vogel D, Rosenthal M, Gogrefe N, Reindl S, Günther S.
J Biol Chem. 2019 Mar 29. pii: jbc.RA118.006973. doi: 10.1074/jbc.RA118.006973. [Epub ahead of print]

Biochemical and structural studies reveal differences and commonalities among cap-snatching endonucleases from segmented negative-strand RNA viruses.
Holm T, Kopicki JD, Busch C, Olschewski S, Rosenthal M, Uetrecht C, Günther S, Reindl S.
J Biol Chem. 2018 Oct 22. pii: jbc.RA118.004373. doi: 10.1074/jbc.RA118.004373.

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.

Funding

Contact

Dr. Maria Rosenthal

Phone: +49 40 42818-942
Fax: +49 40 42818-941
E-Mail: rosenthal@bnitm.de


Scientific Staff

Dr. Dominik Vogel (-942)

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PhD Students

Kristina Meier (-940)
Silke Olschewski (-935)
Efe Can Yildirim (-935)

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Technical staff

Carola Busch (-935)
Nadja Gogrefe (-935)

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Former members

Dr. Tobias Holm
Dr. Sophia Reindl

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Associated Scientists

Dr. Yaiza Fernández García (-942)