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Institut de Biologie StructuraleGrenoble / France

Team Ruigrok

Team Leader: Rob W Ruigrok

Presentation

Replication of negative-sense single-stranded RNA viruses
Negative strand RNA viruses (NSV) have their viral RNA complementary to their mRNA. The viral RNAs are bound to their nucleoprotein to form protein-RNA complexes that are specifically read by a viral RNA-dependent RNA polymerase (RdRp). NSV are classified depending on their genome organization. There are viruses with one long RNA molecule made by several genes, the non-segmented NSV, and viruses with several RNA molecules, the segmented NSV (4). Our team work on influenza virus, a segmented virus, and also on measles virus, a non-segmented virus.

Members

Permanents
Rob Ruigrok, Professor UGA
Thibaut Crépin, CR1 CNRS, HDR
Non-permanents
Amélie Donchet, PhD student
Serafima Guseva, PhD student

Research topics

Measles virus nucleocapsid (Rob Ruigrok)
In 1994 we looked with EM at a sample of the measles nucleoprotein (N) made in insect cells, with a student from the group of Chantal Rabourdin-Combe (Ravanel et al. J Exp Med. 1997, 186(2):269-78). We found that the protein could bind the cellular RNA making nucleocapsids (see figure A). We could see that the N-RNA complex makes a loose helix with the rings easily peeling from the helix. The helical structure determination of measles took a lot of time. First with Guy Schoehn we found that if you cut N with trypsin at residue 405, we observed tight helices that could be used for the first EM structure of the measles nucleocapsid (1). Then, with Malene Jensen and Martin Blackledge we found that the C-terminal part of N makes a flexible, disordered tail (NTAIL), which starts inside the helix and then goes out between the helical turns (3). Therefore, the presence of the flexible NTAIL is the reason that the helix is loose. The first EM structure was done with a Philips CM20 microscope at 200 kV and the images were recorded on film. With Guy Schoen and Irina Gutsche, we then used another microscope, a FEI Polara microscope operated at 300 kV and the micrographs were still on film. With the structure obtained at a resolution of 4.3 Å we could see the protein and also clearly the RNA (9). However, this is still the RNA from the insect cell so the bases of the RNA were blurred. At the same time, Marc Jamin and his student, Filip Yabukarski, found how to make nucleoprotein with a small part of the phosphoprotein of the Nipah virus, very close to measles, and crystallised this protein, the first structure of nucleoprotein of the Paramyxoviruses (6). With this structure, Guillaume Communie, a joint student between Martin and Rob and Sigrid Milles, a postdoc, made the full nucleoprotein with a part of the Phosphoprotein with a TEV site and found that we could bind RNA while the Phosphoprotein dissociated. So we could make N-RNA with the RNA that we could chose as we wanted. We were surprised to see that RNA from the 5’-end of the viral RNA could make nucleocapsids (11). We also found that polyA also makes nucleocapsids but polyU not at all. Now with this discovery we can play with both the protein and the RNA:
-  The EM high resolution structure to see the specific binding of the bases of the RNA
-  The structure of the helix with the full protein
-  The structure of the helix with bits of phosphoprotein bound
-  Change the NTAIL and put another protein or domain for making new vaccines
-  Nucleocapsid formation kinetics by single molecule fluorescence detection


Figure A: EM pictures of measles nucleocapsids. Left the full nucleoprotein and right the capsids after trypsin treatment.

Influenza virus ribonucleoprotein (Thibaut Crépin)
The genome of influenza virus is composed of several RNA molecules encapsidated as individual rod-shaped ribonucleoprotein complexes (RNPs - figure B). Each RNP is made by a viral RNA segment coated with multiple copies of the nucleoprotein (NP) and a polymerase (RdRp) that specifically interacts with both 3’- and 5’- conserved extremities. Each RNP is an independent unit that replicates and transcribes the RNA segment inside the nucleus of the infected cells.


Figure B : influenza virus ribonucleoproteins.

In 1992 we looked with EM at a sample of RNPs directly extracted from the virus with Florence Baudin and Klaus Klumpp. We then started to work with recombinant NP in order to deconvolve both self-oligomerization and RNA interaction of the viral protein. Later, Sylvie Chenavas, a postdoc in the team, showed that NP monomerizes at low salt concentration (5) and we solved the X-ray structure of the monomeric NP (see figure C, left panel). This work was continued by Alice Labaronne, who has started to dissect the specificities of the different types of NP (i.e. NP of influenza A, B, C and D viruses). In parallel to the work on NP, we have also been part of the great adventure on the structural characterization of the heterotrimeric RdRp of influenza virus. This project was a collaboration with the EMBL-Grenoble Outstation and in particular with the groups of Stephen Cusack and Darren Hart. The viral RdRp is an intricate architecture that has long resisted to any recombinant expressions. The first strategy was to identify soluble domains of the viral heterotrimer for further structural analysis. The contribution of the ESPRIT technology (Darren Hart) has been a key element for this purpose. Several isolated domains have been identified (see figure C, right panel) and in particular we have characterized the endonuclease domain of one subunit of the heterotrimeric RdRp (2). After this work, we have tried to express the recombinant heterotrimeric RdRp using a technology developed by Imre Berger (EMBL) in insect cells. Alexandre Monod and Christopher Swale, two PhD students the team, have established the strategy (8, 10) that has culminated in the X-ray structure of the full heterotrimeric RdRp (7).


Figure C : X-Ray structures of influenza RNPs components studied by our team.

The direction followed by the team concerns now the interaction of the RNPs components with the cellular partners involved in the viral replication, like viral RNA splicing and nuclear transport.

Keywords

Negative-sense RNA virus, influenza virus, measles virus, nucleoprotein, nucleocapsid, viral splicing, nuclear transport

Specialized techniques

Production of recombinant proteins and macromolecular complexes
Biochemistry of proteins and RNA
Biophysical characterization of macromolecular complexes
X-ray crystallography
Small-angle X-ray scattering

External collaborations

Imre Berger; University of Bristol
Bernard Delmas; INRA Jouy-en-Josas
Mariette Ducatez, Ecole Nationale Vétérinaire de Toulouse
Denis Gerlier, CIRI, Lyon
Nadia Naffakh, Institut Pasteur
Guido Pintacuda, Centre de RMN à Très Hauts Champs, Lyon
Anny Slama-Schwok, Institut Gustave Roussy

Major publications

1. Schoehn, G., Mavrakis, M., Albertini, A., Wade, R., Hoenger, A. & Ruigrok, R.W.H. (2004). 12 Å Structure of trypsin-treated Measles Virus N-RNA. J. Mol. Biol. 339, 301-312.
2. Dias, A., Bouvier, D., Crepin, T., McCarthy, A.A., Hart, D.J., Baudin, F., Cusack, S. & Ruigrok, R.W.H. (2009). The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458, 914-918.
3. Jensen, M.R., Communie, G., Ribeiro, E.A., Martinez, N., Desfosses, A., Salmon, L., Jamin, M., Mollica, L., Gabel, F., Longhi, S., Ruigrok, R.W.H. & Blackledge, M. (2011). Intrinsic Disorder in Intact Measles Virus Nucleocapsids. Proc. Natl. Acad. Sci. USA 108, 9839-9844.
4. Ruigrok, RWH., Crépin, T. & Kolakofsky, D. (2011). Nucleoproteins and nucleocapsids of negative-strand RNA viruses. Current Opinion in Microbiology 14, 504–510.
5. Chenavas, S., Estrozi, L.F., Slama-Schwok, A., Delmas, B., Di Primo, C., Baudin, F., Li, X, Crépin, T. & Ruigrok, R.W.H. (2013). Monomeric Nucleoprotein of Influenza A Virus. PLoS Pathog 9(3): e1003275. doi:10.1371/journal.ppat.1003275.
6. Yabukarski, F., Lawrence, P., Tarbouriech, N., Bourhis, JM., Delaforge, E., Jensen, MR., Ruigrok, R.W.H., Blackledge, M., Volchkov, V. & Jamin, M. (2014). Structure of Nipah virus unassembled nucleoprotein in complex with its viral chaperone. Nat Struct Mol Biol. 21(9):754-759. doi: 10.1038/nsmb.2868.
7. Reich, S., Guilligay, D., Pflug, A., Malet, H., Berger, I., Crépin, T., Hart, D., Lunardi, T., Nanao, N., Ruigrok, R.W.H. & Cusack, S. (2014). Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature 516, 361–366. doi: 10.1038/nature14009.
8. Crépin T, Swale C, Monod A, Garzoni F, Chaillet M & Berger I. (2015). Polyproteins in structural biology. Curr Opin Struct Biol. 2015 Jun;32:139-46. doi: 10.1016/j.sbi.2015.04.007.
9. Gutsche, I., Desfosses, A., Effantin, G., Ling, W.L., Haupt, M., Ruigrok, R.W.H., Sachse, C. & Schoehn, G. (2015). Near-atomic cryo-EM structure of the helical Measles virus nucleocapsid. Science 348, 704-707. doi: 10.1126/science.aaa5137.
10. Swale, C. Monod, A., Tengo, L., Labaronne, A., Garzoni, F., Bourhis, J.M., Cusack, S., Schoehn, G., Berger, I., Ruigrok, R.W.H. & Crépin T. (2016). Structural characterization of recombinant IAV polymerase reveals a stable complex between viral PA-PB1 heterodimer and host RanBP5. Sci Rep. 6, 24727. doi: 10.1038/srep24727.
11. Milles, S., Jensen, M. R., Communie, G., Maurin, D., Schoehn, G., Ruigrok, R. W. H. & Blackledge, M. (2016). Self-assembly of measles virus nucleocapsid particles: Kinetics and RNA sequence dependence. Angew. Chem. Int. Ed., 55, 9356 –9360. DOI: 10.1002/anie.201602619R1.