STRUCTURAL STUDIES OF THE HIV-1 PRE-INTEGRATION COMPLEX IN/LEDGF INTERACTION INHIBITORS Marc Ruff Laboratory of Integrated structural biology IGBMC, Illkirch, France ruff@igbmc.fr
Schematic diagram of HIV multiplication cycle Pre-integration complex
HIV-1 pre-integration complex PIC: Dynamic complex which composition changes in function of time and space (viral proteins, cellular proteins, DNA ) Integrase: Core protein of the PIC present at all steps: Integrase is the PIC platform protein Enzyme which catalyzes the 3 processing and strand transfer reaction
Integrase flexibility RSV, SIV, PFV, HIV-1 structures show different orientation of the N-terminal domain PFV, HIV1, HIV2 structures show different orientation of the C-terminal domain High flexibility between domains Integrase is a flexible protein (IDP) Protein with multiple structures and multiple functions Partners and/or Post Translational Modifications (PTMs) are needed to stabilize functional conformations Structural and functional studies Isolate stable IN complexes (VBP1, TRN-SR2, LEDGF, INI1) Isolate protein with PTMs (Production in eukaryotic cells)
IN/LEDGF/INI1 : complex formation and purification (E. Coli) LEDGF INI1 HIS-INI1(174-289) His Affinity Gel filtration G200 High salt, detergent Maillot B et al,(2013) PLoS ONE, e60734 + IN/LEDGF High salt, detergent Michel et al,(2009) EMBO J, 28, 980-991 Complex formation by dialysis CryoEM reconstruction and atomic model flexible fitting (NMFF) Gel filtration G200 His-LEDGF 64 kd IN 32 kd Yield: 2.5 mg of complex (INT 0.5L, LEDGF 0.5L, SNF5 1.0L) SNF5 17 kd
Protein produced in E Coli, Insect and mammalian cells Fast rotation: low anisotropy Slow rotation: high anisotropy Fast rotation: low anisotropy Production in mammalian cells: Increase activity, solubility and presence of PTM (phosphorylation and acetylation)
Functional and structural studies: Viral DNA Binding Fast rotation: low anisotropy Slow rotation: high anisotropy IN/LEDGF Maillot et al., PLOSONE, 2013 IN/LEDGF/INI1 Michel et al. EMBO J. 2009 IN/LEDGF complexes, with and without INI1, specifically bind the U5 DNA with similar binding constants
Functional and structural studies: 3 processing and integration Integration 3 processing % GT IN/LEDGF IN/LEDGF/INI1 time INI1 inhibits the 3 processing activity but not integration +td N A IN /LEDGF/vD N A /IN I1 IN /LEDGF/vD N A /td N A integrated p ro ducts -IN I1IB D IN, LEDGF INI1 stabilizes the PIC by maintaining integrase in a stable constrained conformation preventing non-specific interactions and auto integration LEDGF organizes and stabilizes an active integrase tetramer suitable for specific vdna integration.
IN/LEDGF/vDNA complex IN Cter IN tetramer Viral DNA IN Nter LEDGF dimer
Response (RU) 12 10 8 6 4 2 0-2 5.0 µm 2.5 µm 1.3 µm 0.63 µm 0.31 µm 0.16 µm 78 nm 39 nm 20 nm 9.8 nm 0 100 200 300 400 Time (s) 1- Transfection of 293T cells 2- Addition of 3- Supernatant collection pnl4-3 DNA compounds N pnl4-3 IN T174I DNA N 2h N DMSO 0.5% SQV 50 nm (5x EC50) RAL 50 nm (5x EC50) Mut101 2.5 µm (5x EC50) Mut063 5 µm Non active drug N 48h Response (RU) 12 10 8 6 4 2 0-2 Quantification of p24 antigen Infection of MT4 5.0 µm 2.5 µm 1.3 µm 0 100 200 300 400 Time (s) RLU 80 60 40 20 0 L 1 NL4-3 wt NL4-3 IN T174I 3 % CCD-IBD Inhibition EC 50 HxB2 (µm) 100 80 60 40 20 0 0.001 0.01 0.1 1 10 100 Concentration (µm) 100 10 1 0.1 0.1 1 10 100 IC 50 CCD-IBD (µm) % IN-LEDGF Inhibition EC 50 HxB2 (µm) 100 80 60 40 20 0 0.001 0.01 0.1 1 10 100 Concentration (µm) 100 10 1 0.1 0.1 1 10 100 IC 50 IN-LEDGF (µm) Mut101 IN-LEDGF IC 50 (nm) % IN-LEDGF Inhibition 100 80 60 40 20 0 0.001 0.01 0.1 1 10 100 Mut101 concentration (µm) 800 600 400 200 0 [LEDGF] 480 nm 0 100 200 300 400 500 LEDGF concentration (nm) Protein Protein interaction and allosteric inhibitors HTP IN LEDGF interaction inhibitors screening (HTRF) IN CCD Production and crystallization Inhibitors crystal soaking, data collection, structure Inhibition of integration and viral maturation in late phase Allosteric Inhibitor Structure based drug design in-vitro functional assay A B Mut029 Mut047 Mut049 Mut062 Mut075 Mut101 C D Mut029 Mut047 Mut049 Mut062 Mut075 Mut101 E F 7.5 nm 15 nm 30 nm 60 nm 120 nm 240 nm In cellulo functional assay A C 14 [Mut101] B 14 [Mut101] D
IN LEDGF interaction and IN allosteric inhibitors INLAIs
Structure comparison of HIV-1 Integrase Catalytic core Domain with and without MUT101 A C E B D D F C
INLAIs inhibitors Summary ~70 structures solved IN/LEDGF interaction and allosteric inhibitors. Ligand binding lead to structural changes in the active site, dimeric interface, accessible surface. Mammalian cells production: increase integrase stability, solubility and 3 processing activity. Presence of acetylation and phosphorylation LEDGF stabilizes an IN tetramer and increases its integration and 3 processing activity Integrase VBP1 TRN-SR2 Integrase LEDGF TRN-SR2 Integrase LEDGF Integrase LEDGF DNA INI1 prevents non specific aggregation and auto integration on the way to nucleosomes in the nucleus Integrase LEDGF INI1 3 PROCESSING INTEGRATION Integrase LEDGF Nucleosome
Acknowledgments Integrative Structural Biology, IGBMC, Illkirch Sylvia Eiler Nicolas Levy Karine Pradeau Robert Drillien Damola Oladosu Faculty of pharmacy, University of Strasbourg, Illkirch Yves Mely Institute of cellular and molecular Biology, Strasbourg, Sylviane Muller, François Stricher Group of Molecular Electron Microscopy, IGBMC, Illkirch Patrick Schultz Corinne Crucifix CovalX, Technoparkstrasse, 1, CH-8005, Zürich, Zwitzerland Alexis Nazabal Mutabilis, France Richard Benarous, François Moreau Infection Diseases Department, Cochin Institute, Paris Stéphane Emiliani Fundamental Microbiology and Pathogenicity, University of Bordeaux 2 Vincent Parissi ENS, CNRS, Pasteur Institute, Lyon Marc Lavigne Functional Genomics and Cancer, IGBMC, Illkirch Ali Hamiche Catherine Ramain Bio-organic Mass Spectrometry Laboratory, University of Strasbourg, Cronembourg Sarah Sanglier-Cianferani Sarah Lennon Members of the IGBMC Mass Spectrometry Facility Members of Structural Biology and Genomics platform CEGBS, Illkirch Members of the bioinformatic group and platform Members of the imaging platform Members of the IGBMC s common services Cell, baculovirus and cloning facilities