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Structural And Functional Studies Of Retroviral Pre-Integration Complexes

Reference : PhD Marc RUFF

Offer publication : April 6, 2016

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 pre-integration 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. 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 rise 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. The strategy for the production of stable complexes has been developed in the laboratory (Levy et al. Nat. Commu., 2016, in press). Stable complexes production is done in two organisms, E. Coli 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, X-ray crystallography 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.

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Application Deadline : Dec. 31, 2016

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