Institut de Biologie StructuraleGrenoble / France


Bacteriophage structure and life cycle

Team Members : Maria Bacia, Grégory Effantin, Guy Schoehn
Collaborations : Cécile Breyton (IBS), Rob Lavigne (Belgique) ; Takashi Yamada (Japon)

We would like to further develop our studies on phages, a subject in which we have collaboration with an IBS group (C Breyton) and also groups in Paris, Japan and Belgium. Electron microscopy is a particularly suitable tool to study the phage life cycle, which will make full use of the instruments and expertise of the group. Classical cryo-EM coupled with image analysis, tomography of bacteria infected by the phages with cellular EM techniques (CEMOVIS for example), and other techniques will be employed to address different aspects of the phage life cycle.

Effantin G, Hamasaki R, Kawasaki T, Bacia M, Moriscot C, Weissenhorn W, Yamada T, Schoehn G. (2013). Cryo-electron microscopy three-dimensional structure of the jumbo phage ΦRSL1 infecting the phytopathogen Ralstonia solanacearum. Structure 21(2):298-305.

The head and the helical tail of this phage have been solved to nanometer resolution. This result is, to our knowledge, the highest available resolution for a phage tail. It is also the highest resolution achieved for the baseplate and the head of a jumbo phage. These 3D structures also confirm that the jumbo phage share the same fold as other phages (at least for the head and the helical tail).

Structure and life cycle of Foamy virus

Team Members: Grégory Effantin, Léandro Estrozi, Guy Schoehn
Collaborations : EBEV group IBS (Responsable Winfried Weissenhorn)

Foamy viruses belong to retrovirus family (enclosing VIH). They are responsible of persistent infections in a lot of animal species without causing direct illness. For this reason they are good candidate for gene therapy.
In order to understand their structure, we have characterized the Prototype Foamy Virus (PFV) by cryo-electron microscopy and cryo-electron tomography using the Polara electron microscope with its K2 direct electron detection camera. The mature and infectious form of PFV has a complex structure : a nucleocapsid at the core level and a dense glycoproteins network inserted into the viral membrane. These last ones are trimeric and may assemble in hexagonal networks. The in situ structure of the glycoprotein at 9 Å of resolution has showed a superhelix conformation of one part (which is typical for fusion proteins of class I retrovirus), and six transmembrane proteins helices. These results allow a better understanding of the life cycle of PFV.

3D reconstruction obtained by cryo-electron microscopy of hexagonal assembly composed by 6 trimers of the PFV glycoprotein. They are in pink and blue, the viral membrane is in light grey.

Influenza Virus

Team Members: Guy Schoehn
Collaboration : Viral Replication Machines group IBS (Rob Ruigrok, Thibaut Crépin)

Since several years we collaborate with the team of Pr Rob Ruigrok (IBS) to decipher from a structural point of view different components of the flu virus. We are mainly interested in polymerase and nucleoproteins of this virus.

References :
Labaronne A, Swale C, Monod A, Schoehn G, Crépin T, Ruigrok RW. (2016). Binding of RNA by the Nucleoproteins of Influenza Viruses A and B. Viruses. 8(9). pii: E247.
Swale C, Monod A, Tengo L, Labaronne A, Garzoni F, Bourhis JM, Cusack S, Schoehn G, Berger I, Ruigrok RW, 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.

Nucleocapsid of the Measle Virus

Team Members : Grégory Effantin, Wai-Li Ling, Guy Schoehn
Collaborations : Gutsche Team, Groupe MEM IBS ; Viral Replication Machines group (Rob Ruigrok) ; Protein Dynamics and Flexibility by NMR Group (M.Blackledge)

Measle virus is an envelopped RNA virus. This single strand RNA is protected by a nucleoprotein within the viral particle. The nucleoprotein of this virus has been expressed in insect cells and during the expression, proteins are associated with cellular RNA to form pseudo-nucleocapsids.
These nucleocapsids have been visualized by cryo-electron microscopy in their native state after rapid freezing in liquid ethane and using the Polara microscope. The images have been collected on films and analysed in collaboration with EMBL at Heidelberg. These images have allowed to calculate the 3D structure of these nucleocapsids at the amazing resolution of 4.3 Å (Gutsche et al., 2015).
With the new camera (electron counting direct detection camera), reachable resolutions with this microscope should still improve.

3D reconstruction at 4.3Å resolution of the nucleocapsid of the measle virus. RNA is in green.

References :
Milles S, Jensen MR, Communie G, Maurin D, Schoehn G, Ruigrok RW, Blackledge M. (2016). Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence. Angew Chem Int Ed Engl. 2016 Aug 1;55(32):9356-60.
Gutsche I, Desfosses A, Effantin G, Ling WL, Haupt M, Ruigrok RW, Sachse C, Schoehn G. (2015).Structural virology. Near-atomic cryo-EM structure of the helical measles virus nucleocapsid. Science. 348(6235):704-7.