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2014

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Structural and functional studies of retroviral integrases

Research unit

UMR 7104 - Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)
1 rue Laurent Fries 67404 ILLKIRCH

Group

Name: Expression de l'information génétique

Group leader: MORAS Dino - moras@igbmc.fr

Group leader's phone: 0388653220

Website: Visit website

Group organization:
- Chercheurs: 4
- ITA: 3
- Doctorants: 2
- Post-Docs: 3
- Autres: 1

Publications of the team linked to the topic (3 last years):
1) Le Rouzic E, Bonnard D, Chasset S, Bruneau JM, Chevreuil F, Le Strat F, Nguyen J, Beauvoir R, Amadori C Brias J, Vomscheid S, Eiler S, Levy N, Delelis O, Deprez E, Saïb A, Zamborlini A, Emiliani S, Ruff M, Ledoussal B, Moreau F, Benarous R. Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage. Retrovirology. 2013 Nov 21;10(1):144.
2) Maillot, B., Lévy, N., Eiler, S., Crucifix, C. , Granger, F., Richert, L., Didier, P., Godet, J., Pradeau-Aubreton, K., Emiliani, S., Nazabal, A., Lesbats, P., Parissi, V., Mely, Y., Moras, D., Schultz, P. and Ruff, M., (2013), Structural and functional role of INI1 and LEDGF in the HIV-1 preintegration complex, PLoS ONE 8(4): e60734.
3) Cribier A, Segeral E, Delelis O, Parissi V, Simon A, Ruff M, Benarous R, Emiliani S. (2011). Mutations affecting interaction of integrase with TNPO3 do not prevent HIV-1 cDNA nuclear import. Retrovirology, 104, doi: 10.1186/1742-4690-8-104

About PhD

PhD Director: RUFF Marc - ruff@igbmc.fr

Phone: 0388653349

Junior advisor: LEVY Nicolas

Co-tutely: non

Co-Director: non

About PhD topic :

Title: Structural and functional studies of retroviral integrases

Project: An essential step in retroviral infection like HIV is the integration of the viral genome into the host cell chromosomes. The integrase protein is the platform protein present in the different steps of retroviral infection involving the preintegration complex (PIC). Integrase is required for reverse transcription, PIC migration along microtubules, transfer to the nucleus, chromatin targeting and integration. Retroviral integrase is a flexible protein showing high inter-domain flexibility, and thus belongs to the growing family of intrinsically disordered proteins. This intrinsic disorder explains the integrase ability to interact with multiple partners and allows its multiple biological functions. In this project we will compare by in-vitro and in-cellulo structural and functional studies, several retroviral integrases alone or in complex with DNA and cellular and viral cofactors to understand the structure-function relationships underlying the different roles of the integrase viral protein.
The different functions of integrase in viral replication are associated with different structural conformations which could be induced by the proteins, DNA, RNA partners molecules as well as by post-translational modifications like acetylation, methylation and phosphorylation. We will screen the different complexes and proteins for their stability and solubility which are needed for structural and functional studies.
The main objective of the proposed project is the structural and functional analyses of these complexes. The techniques used in the laboratory go from cloning to structural studies through protein production, physico-chemical and functional characterization.
The first results obtained in our laboratory concerned the HIV-1 integrase in complex with the cellular protein LEDGF (Michel et al., EMBO J, Feb 2009). A structure done by cryo-electron microscopy of this complex has been solved at 14 Ǻ resolution and gave raise to the first known model for viral DNA integration in the cellular DNA. A second complex of integrase in interaction with two cellular proteins (LEDGF, INI1) has been solved by cryo-electron microscopy (Maillot et al., PloSone, 2013). The first step in this project will be the development of large-scale recombinant protein production for the complexes. Stable complexes production is done in three organisms, E. Coli, insect cells and mammalian cells. Purification is done by affinity chromatography followed by gel filtration, and depending of the cases by another stage of purification (hydrophobic interaction, ion exchange).
Crosswise, structural and physicochemical characterization techniques are used (ES-MS, Trypsin digestion /MALDI, High Mass MALDI-TOF, NMR, electron microscopy, ultra analytical centrifugation). The functional characterization is done by analyzing the capacity of the complexes to fix the viral DNA by fluorescence anisotropy and correlation techniques.
Structural studies are made with various techniques: electron microscopy for the characterization and determination of structures to low resolution, radiocristallography for determining high-resolution structures (synchrotron for data collection and robots for validation of crystals and the automatic acquisition of diffraction data), small angles X-Ray scattering (SAXS) for low resolution solution structures.

Wished skills: Structural and molecular biology, chemistry, biophysics, biochemistry

Expertises which will be acquired during the training: Production and purification of multi-protein complexes using prokaryote and eukaryote expression systems. Biochemical and biophysical complex characterization. Structure determination by cryo-EM and crystallography, structure function relationship studies.