Burmeister team

Responsible : Wim Burmeister

We work on the structure and the function of viral proteins, in particular of the vaccinia virus replication machinery combining structural biology (X-ray crystallography, electron microscopy, small-angle X-ray scattering) with biophysical techniques (MALLS, surface plasmon resonance, fluorescence anisotropy).
One of the great success stories of modern medicine has been the eradication of smallpox virus declared in 1979 after a long vaccination campaign with vaccinia virus (VV). After the terrorist attacks of 2001, new interest in poxviruses arose in the context of a potential use by bioterrorism. Finally, with a vaccination coverage of the human population which is almost non-existent, nowadays the principal risk is the introduction of an orthopoxvirus into the human population from an animal reservoir. It became obvious that poxvirus circulate widely at the level of farm animals and wild rodents. Recently, several diseases with the risk of an epidemic arose after the introduction of a virus from an animal reservoir (HIV, SARS, bird flu, Ebola virus). Thus it seems essential to be prepared for poxvirus infections by the availability of a panel of antivirals ; but, at the current date, there are only two molecules, brincidofovir and tecovirimat. Better knowledge of the structure of poxvirus proteins in general and of the polymerase in particular will allow to develop new compounds and to understand better resistance mechanisms against the current molecules. We work on vaccinia virus which is 98 % identical to smallpox virus at the amino acid level of the replication proteins.
We concentrate on the essential replication proteins, the helicase-primase D5, the DNA polymerase composed of the catalytic subunit E9 and the processivity factor composed of the accessory protein A20 and the uracil N-glycosylase D4, whose DNA binding capacity is used. During the last years, we obtained structural information at increasing resolution about the tridimensional structure of these proteins which is filling progressively our initial low-resolution envelope (Figure 1, [6]).

Figure 1 : Model of the polymerase holoenzyme complex. The envelope has been obtained by SAXS [6]. The available high-resolution structures have been placed. The DNA bound to the polymerase could be modelled [1] whereas the DNA bound to D4 has been observed in a complex structure [5]. The overall organization of the replication fork remains unknown.

Due to dynamics and flexibility, the high resolution structure of the helicase primase D5 is elusive. Low-resolution on the domain structure has been obtained [2], which relates it closely to the Lta helicase of polyomavirus. The protein has been extensively studied by small-angle X-ray scattering (SAXS) leading to new methodological developments. With the rapid evolution of cryo-electron microscopy at IBS, we hope to be able to obtain new functional and structural information in the near future.
In contrast, with the 2.7 Å structure of the catalytic polymerase subunit E9 (Figure 2, [1]) a breakthrough has been obtained.

Figure 2 : Domain organization of the E9 polymerase

Based on these results, we work on the function of the replication machinery which replicates the unique DNA structure of the poxvirus genome which consists of a linear double-stranded DNA which is circularized at the ends. The detailed knowledge of the interfaces of the different subunits of the polymerase will be used for the design of inhibitors which interfere with the assembly of the protein complex. This aspect is developed in the context of an FRM (Fondation de Recherche Médicale) - funded project on the optimization of peptides by phage display aiming at the disruption of interaction surfaces of proteins of the replication machineries. In particular, interface between the D4 uracil-DNA-N-glycosylase subunit and the A20 protein of the processivity factor will be targeted which has already been studied extensively (Figure 3, [3,5]).

Figure 3 : Part of the A20 (pink) - D4 (green) interface

This project is based on a collaboration with Frederic Iseni who directs the Laboratory of Virology at the IRBA, Bretigny-sur-Orge in the Paris area.

Keywords

poxvirus, DNA replication, DNA polymerase, helicase, primase, processivity factor

Specialized techniques

• Recombinant protein production in insect cells and E. coli
• X-ray crystallography
• Biochemical and biophysical characterization (Fluorescence anisotropy, Surface plasmon resonance, SAXS, circular dichroïsm, MALLS)
• Electron microscopy in collaboration with the Electron Microscopy and Methods group.

Members of the team

• Wim Burmeister, Professor
• Nicolas Tarbouriech, Lecturer, Maître de conférence

Major Publications

[1] Tarbouriech N, Ducournau C, Hutin S, Mas PJ, Man P, Forest E, Hart DJ, Peyrefitte CN, Burmeister WP, Iseni F. The vaccinia virus DNA polymerase structure provides insights into the mode of processivity factor binding. Nat Commun 8:1455 (2017). link.

[2] Hutin, S., Ling, W. L., Round, A., Effantin, G., Reich, S., Iseni, F., Tarbouriech, N., Schoehn, G. & Burmeister, W. P. Domain organization of vaccinia virus helicase-primase D5. J. Virol. 90, 4604-13. doi : 10.1128/JVI.00044-16. (2016). link

[3] Contesto-Richefeu, C., Tarbouriech, N., Brazzolotto, X., Burmeister, W.P., Peyrefitte, C.N. & Iseni, F. Structural analysis of point mutations at the Vaccinia virus A20/D4 interface. Acta Cryst. F72, doi:10.1107/S2053230X16011778 (2015). link

[4] Thierry, E., Brennich M., Round, A., Buisson M., Burmeister, W. P. & Hutin, S. Production and characterization of Epstein-Barr virus helicase primase complex and its accessory protein BBLF2/3. Virus Genes. doi 10.1007/s11262-015-1233-6 (2015). link

[5] Burmeister, W.P., Tarbouriech, N., Fender, P., Contesto-Richefeu, C., Peyrefitte, C.N. & Iseni, F. Crystal structure of the vaccinia virus uracil DNA-glycosylase in complex with DNA. J Biol. Chem. pii : jbc.M115.648352. (2015). link

[6] Sèle, C., Gabel, F., Gutsche, I., Ivanov, I., Burmeister, W.P., Iseni, F. & Tarbouriech, N. Low-Resolution Structure of the Vaccinia Virus DNA Replication machinery. J. Virol. 87 : 1679-1689 (2012). link

Contact for the webpage : wim.burmeister(at)ibs.fr