Cryo-EM structure of the deltaretroviral intasome in complex with the PP2A regulatory subunit B56γ

Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.

and integrated in Xia2 3 using XDS 4 , which identified datasets belonging to two unique space groups. Datasets were scaled and merged in CCP4i2 5 Aimless 6 , with the resolution limit adjusted accordingly until satisfactory signal-to-noise and completeness were reached. Unit cell composition was estimated from Matthew's coefficient. Phases were obtained by molecular replacement in PHASER 7 through the PHENIX software suite 8 . The mouse mammary tumour virus (MMTV) IN/CCD (PDB ID: 5CZ1) was used as a search model and resulted in a solution with a TFZ score of 50. PHENIX Autobuild 9 was successful in building the majority of residues in all chains. The remaining residues were added manually in Coot and the model was refined in Refmac 5.8.

Crystallisation of HTLV-1 IN/CTD (228-286)
The HTLV-1 IN/CTD construct that led to good quality highly diffracting crystals encodes for residues 228-286 and is further referred to as the IN/CTD. Expression was conducted as described above for HTLV-2 IN/CCD. Extraction was performed in 50 mM Tris-HCl pH 7.4, 0.5 M NaCl, 10 mM imidazole, 1 mM PMSF, through sonication of the resuspended. This was then clarified by centrifugation at 50 000 g for 15 min at 4ºC. Once bound to the IMAC resin, 1 M ammonium tartrate dibasic as the optimal condition for crystal growth in the hangingdrop format, and the protein:precipitant ratio of 2:1. Apparent over-nucleation, affecting crystal size, was resolved by microseeding. Seeds were prepared by maceration of medium-size crystals in mother liquor containing the same crystallisation components as the target condition. Seeds were then collected, diluted 1,000-fold in the reservoir solution and kept on ice. Streak-seeding was performed immediately after.
Crystals were harvested and cryoprotected in a step-wise fashion with reservoir solution supplemented with 10%, then 20% glycerol (v/v). Crystals were then cryo-cooled in liquid nitrogen. Native data collection was carried out at beamline I04 at Diamond Light Source (Didcot, UK). A 0.979 Å wavelength beam was used to collect diffraction images with 0.01 s exposures per 0.1 o oscillation angle, over a total rotation angle of 360 o . Crystals diffracted to a maximum resolution of 1.14 Å. Data were indexed and integrated in XDS 4 via Xia2 3 . Datasets were scaled and merged in CCP4i2 5 using Aimless 6 , with the resolution limit adjusted accordingly until satisfactory signal-to-noise and completeness were reached. Unit cell composition was estimated from Matthew's coefficient. Phases were obtained by molecular replacement using PHASER 7 through the PHENIX software suite 8 . Using the MVV IN/CTD structure as a replacement model (PDB code 5LLJ) resulted in a single solution with a TFZ score of 30.1. PHENIX Autobuild 9 was successful in building the majority of residues in all chains. The remaining residues were added manually in Coot and the model was refined in Refmac 5.8.

Crystallisation of HTLV-1 IN (200-297) : B56γ complex
The PP2A regulatory subunit B56γ (11-380) (further referred to as B56g) was expressed and purified as described previously 10  Data were collected on beamline I24 at Diamond Light Source (Didcot, UK). Reflections were indexed using Xia2 3 in 3dii mode 4 . In order to increase the signal-to-noise ratio, two wedges of images with the highest Bragg reflection number were extracted from the original dataset and re-indexed together in space group P43212. Phaser 7 was used for phasing by molecular replacement using the B56γ structure (PDB ID: 2JAK) as the template. PHENIX Autobuild 9 was successful in building the majority of residues in all chains. The remaining residues were added manually in Coot and the final model was refined in Refmac 5.8.

Tissue culture and Flag-immunoprecipitation (IP)
Human Embryonic Kidney SV40 large T cells (further referred to as 293T) were cultured in Dulbecco's Modified Eagle Medium (Sigma) supplemented with 10% foetal bovine serum (Sigma), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Sigma) and grown in a humidified incubator at 37°C with 5% CO2. 293T cells were transfected with 20 µg plasmid DNA by calcium phosphate precipitation as previously described 11