B′-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase

To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B′ family of the regulatory subunits. B′-PP2A and HTLV-1 IN display nuclear co-localization, and the B′ subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein–protein interaction interface maps to a patch of highly conserved residues on B′, which when mutated render B′ incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity.

The prokaryotic expression construct for His 6 -TEV-B'(11-380) (PDB ID: 2JAK) (28) was obtained from Source Bioscience UK Limited and provided from the University of Oxford Structural Genetics Consortium (Stockholm clone name PPP2R5CA-c005). Constructs used to express C-terminally His 6 -tagged HTLV-1 IN was described previously (56), and pHTLV-2 IN-His 6 was kindly provided by Peter Cherepanov (Cancer Research UK, Clare Hall Laboratories). Unless stated otherwise, all ORFs were amplified using a HeLa cDNA library (Clontech) as template. pCDF-H6P-PPP2CA was generated by amplifying the PPP2CA gene using primers GM93 and GM95 followed by amplification of this PCR product with primers GM94. This produces the following sequence His 6 -BamHI-HRV 3C-EcoRI-PPP2CA-STOP-SalI-HindIII-NotI which allows the expression of an N-terminally His 6 -fused protein of which the His 6 -tag can be removed by Human Rhinovirus (HRV) 3C digestion. The B'' ORF was amplified using primers GNM278 and GNM279 and cloned in between BamHI/SalI restriction site of pGEX-6P1 (GE Healthcare), giving pGM-GST-B''. pCDF-H6P-  and pCDF-  were generated using the Stockholm clone PPP2R5CA-c005 (28) as a template with primers GM142 and GNM276 for deletion mutant B'  and GNM271 and GM143 for deletion mutant B' . The amplicons were digested with EcoRI/SalI (B'(11-194)) or EcoRI/NotI (B'(195-380)) and cloned in between the respective restriction sites of pCDF-H6P-PPP2CA. pET28a-SUMO-PPP2R5D(76-501) was amplified using GM112 and GM55 and ligated in between the BamHI/SalI restriction sites of pET28aSUMO (kindly given by Dr. Andre Ambrosio). For the expression of the  isoform of the scaffolding subunit, PPP2R1A was amplified using primers GM103 and GM97 and cloned in between the BamHI/XhoI restriction sites of pET28aSUMO. pCDF-H6P-PPP2R5E(51-401) was generated by ligating the amplicon made with primers GNM315 and GNM316 and digested with MfeI and SalI into EcoRI/SalI digested pCDF-H6P-PPP2CA. The HTLV-1 IN synthetic gene was amplified from pQHTLV-1 IN S -Flag (15) using primers GM109 and GM110 and ligated into EcoRI/SalI digested pET28aSUMO, giving pET28aSUMO-HTLV-1 IN s . The HTLV-2 IN ORF was amplified using pHTLV-2 IN-His 6 as a template with primers GM144 and GM131. This amplicon was digested with MfeI/SalI and ligated into EcoRI/SalI digested pET28aSUMO. All B'(11-380) point mutants described were sub-cloned into pET28a-SUMO to express as His 6 -SUMO fusions to produce recombinant protein. All plasmids were sequence verified.

Protein purification
For expression of HTLV-1 IN-His 6 , HTLV-2 IN-His 6 , HIV-1 IN-His 6 , FIV IN-His 6 , and GST-B'', the corresponding prokaryotic expression plasmids were transformed into the PC2 strain (22). Bacteria were grown in Luria Bertani medium at 30°C until an OD 600nm of 0.9 was reached. The temperature was reduced to 25°C and protein expression was induced by addition of 0.01% IPTG. Four hours later, bacterial pellets were collected and stored at -80°C until use. All other recombinant proteins were expressed in the Rosetta2(DE3)pLacI strain (Novagen) and grown in Terrific Broth. Transformed bacteria grown in the appropriate selective media were allowed to reach an OD 600nm of 2.5-3 upon which the temperature was reduced to 25°C and protein expression induced by addition of 0.01% IPTG. Four hours later the bacteria were collected by centrifugation and pellets were frozen at -80°C until further use. All further procedures were done on ice or at 4°C. To purify the IN-His 6 proteins, bacterial pellets were thawed, resuspended and sonicated in core buffer (50 mM Tris pH7.4, 1 M NaCl, 7.5 mM CHAPS) supplemented with 1 mM PMSF. Cellular debris was removed by centrifugation at 50 000 g. Supernatant was supplemented with 10 mM imidazole and bound to HisSelect resin (Sigma). After extensive washes in wash buffer (core + 10 mM imidazole), IN proteins were eluted in 10 1 ml fractions with elution buffer (core buffer + 200 mM imidazole). Positive fractions were pooled, supplemented with 5 mM DTT, concentrated and supplemented with 10% glycerol final concentration, aliquoted and snap frozen in N 2 (l).
To produce untagged IN proteins, the His 6 -SUMO-HTLV-1 and 2 IN proteins eluted from the HisSelect column were supplemented with 5 mM DTT and Ulp1 sumo-protease to remove the His 6 -SUMO tag. Digestion was done overnight at 4°C. Cleaved protein was then diluted four fold in ice cold buffer A (25 mM Tris pH7.4, 7.5 mM CHAPS) before binding to an SP sepharose column (GE Healthcare) equilibrated with 25 mM Tris pH7.4, 250 mM NaCl, 7.5 mM CHAPS. After extensive washes, untagged HTLV-1 or 2 IN was eluted by applying a linear NaCl gradient. Positive fractions were pooled and further purified by size exclusion chromatography (HiLoad 16/60 SD200 column) in 25 mM Tris pH7.4, 1 M NaCl, 7.5 mM CHAPS. Positive fractions were pooled, supplemented with 5 mM DTT, concentrated and snap frozen in N 2 (l). Purification of B'(11-380) was done as previously described (28).
Bacterial pellets were resuspended in 25 mM TrisHCl pH7.4, 0.5 M NaCl, 1 mM PMSF, supplemented with 0.1 mg/ml lysozyme, sonicated and the soluble fraction was bound to HisSelect. After extensive washes in the used sonication buffer supplemented with 10 mM imidazole, His 6 -SUMO-B'(11-380) point mutants were eluted by increasing the imidazole concentration to 200 mM. Positive fractions were pooled, supplemented with 5 mM DTT and the His 6 -SUMO tag was cleaved off by Ulp1 protease treatment overnight at 4°C. Following a 5 fold dilution of the cleaved protein in 25 mM Tris pH7.4, the proteins were purified as wild type B'(11-380). His 6 -SUMO-A was expressed and purified as the His 6 -SUMO-B'(11-380) point mutants, with the exception that size exclusion chromatography of the untagged protein was done in ice cold 25 mM Tris pH7.4, 500 mM NaCl. B'(76-501) was expressed as a His 6 -SUMO fusion protein. The bacterial pellets were resuspended in 25 mM Tris pH7.4, 150 mM NaCl, 1 mM PMSF. After sonication, the soluble supernatant was bound to HisSelect, after elution the His 6 -SUMO-tag was removed by Ulp1 cleavage overnight. The untagged protein was then purified by size exclusion in 25 mM Tris pH7.4, 150 mM NaCl.
Positive fractions were pooled, supplemented with 5 mM DTT, concentrated and flash frozen in N 2 (l). GST-B'' was extracted from the bacterial pellets in 25 mM Tris pH8, 100 mM NaCl, 1% TX-100, 1 mM CaCl 2 , 1 mM PMSF. Following sonication and removal of debris by centrifugation, GST-B'' was allowed to bind glutathione sepharose (GE Healthcare). After extensive washes B'' was released from the beads by HRV 3C protease digestion overnight.
Untagged B'' was further purified by anion exchange chromatography (linear NaCl gradient from 50 mM to 500 mM). Positive fractions were pooled, supplemented with 2 mM DTT, concentrated and flash frozen in N 2 (l).

Tissue culture, stable cell lines and immunostaining
HEK293T and HeLa cell lines were maintained in Dulbecco's Modified Eagle Medium (Sigma) supplemented with 10% fetal bovine serum (Sigma), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Sigma). The HEK293T cell line stably expressing Flag-tagged HIV-1 IN s was published previously (57), and was maintained in 300 g/ml hygromycin B supplemented medium. Retroviral particles were produced as described previously (55).
Fourty eight h post-infection the cells were selected with 0.5 g/ml puromycin.
For immunostaining, HeLa cells were plated out in 8-well Lab-Tek II Chamber slides (Nunc) to reach 80% confluence the next day. HeLa cells were transfected using 150 ng of plasmid DNA in total by X-tremeGENE 9 transfection reagent (Roche) following manufacturer's instructions. Twenty h post-transfection the cells were fixed for 10 min in 4% paraformaldehyde (diluted in phosphate buffered saline (PBS)) followed by permeabilization using 0.1% Triton X-100 diluted in PBS. All antibodies were diluted in blocking buffer (10% FBS, 20 mM NH 4 Cl in PBS). Flag-tagged IN proteins were detected using the monoclonal M2 anti-Flag antibody (Sigma, 1:500), and EGFP-B'(11-380) was detected using the rabbit anti-EGFP antibody (Life Technologies, 1:2000). Goat anti-mouse IgG conjugated to Texas Red (Life Technologies) and Alexa 488 conjugated goat anti-rabbit IgG (Life Technologies) were diluted 1:400. DNA was visualized by 4',6-diamidino-2-phenylindole (DAPI, Life Technologies) staining. Images were acquired using an Olympus microscope with a 60x Plapon oil objective (NA 1.4). DAPI was excited with a 405 nm laser beam, whilst 488 nm, respectively 559 nm laser beams were used to excite the Alexa 488 and TexasRed dyes.
Images were acquired sequentially at 40 s/pixel, 1024x1024 image resolution.
To make the extracts, the resuspensions were allowed to thaw fast at 37°C and immediately centrifuged at 16 000 g, 30 min at 4°C. Supernatants were collected, supplemented with 0.1 M NaCl, 0.5 % Nonidet P-40 (NP-40) and 0.5 mM PMSF. The extracts were pre-cleared over 100 l washed Protein G agarose (GE Healthcare) followed by binding to 100 l anti-Flag agarose (Sigma). Flag-tagged protein complexes were allowed to bind to the beads for 3h by end-over-end rocking at 4°C. Beads were washed extensively with wash buffer (FTB supplemented with 0.5 % NP-40, 0.1 M NaCl) and bound proteins were eluted with 0.04 mg/ml Flag peptide (Sigma) in wash buffer. Eluted proteins were precipitated by trichloric acid, pellets were dissolved in SDS loading buffer and proteins were separated on a 4-20% BisTris gel (Life Technologies). The gels were stained in Colloidal Coomassie (Sigma) and bands were excised and sent for tandem mass spectrometry analysis to the Taplin Mass Spectrometry Facility. The MS data was analyzed by the Taplin Mass Spectrometry facility and Sequest was used to search data. The data was filtered based on XCorr and dCn values and then manually inspected for proteins that only had three or few peptide matches. Values of 1.5 for peptides with one or two charges, 3.0 for three charges for XCorr and 0.1 or higher for dCn were used. The data was also searched allowing for either partial tryptic peptides or with no enzyme specificity and required that all peptides be tryptic. Only proteins with minimally 2 unique peptide matches, and that were absent in the negative control sample are listed in Supplementary Tables S2 and S3. For small scale IPs, 293T cells grown in 1 10 cm dish, were harvested by trypsinization and washed in ice cold PBS. All procedures were done on ice or at 4°C. Cells were lysed in 5 volumes of IP buffer (10 mM TrisHCl pH7.5, 150 mM NaCl, 10% glycerol, 1% NP-40, 2 mM MgCl 2 , Complete EDTA free (Roche), 2 mM DTT), left on ice for 10 min and cellular debris was removed by centrifugation at 16 000 g for 30 min. To verify the binding between Flagtagged B'(11-380) and the scaffold and catalytic subunit, IP buffer without detergent was used. Supernatants were allowed to bind to 25 l pre-washed anti-Flag agarose beads (Sigma) by end-over-end rocking at 4°C. Beads were washed 4 times in 1 ml of IP buffer.
After removing all remaining liquid from the beads, proteins were eluted by boiling the beads in 45 l of Laemmli buffer. After separation of the proteins on an 11% SDS-PAGE denaturing gel, proteins were electrotransferred onto nitrocellulose membrane. Blots were blocked in 5% milk/PBS and probed with the following antibodies: horse radish peroxidase (

Phosphatase assays
To isolate B'-PP2A holo-enzymes from mammalian cells, a HEK293T cell line was generated that stably expresses full-length wild type Flag-B'by retroviral transduction as described above. The cell line was maintained in 0.5 g/ml puromycin. Flag-B'-PP2A holoenzymes were purified as described previously (31) and verified by gel and western blot to confirm the presence of all three subunits. To quantify the amount of Flag-B'-PP2A purified, 10 l was separated on an 11% SDS-PAGE gel next to a dilution series of BSA. Following silver staining, using ImageJ it was estimated that the concentration of holo-enzyme in our eluate was 28 nM. The colorimetric malachite green phosphatase assay was used to measure PP2A enzymatic activity using the PP2A specific phospho-Threonine peptide (K-R-pT-I-R-R) as a substrate. Absorbance was read at 620nm. A standard curve was made using a dilution series of potassium phosphate ranging from 0 to 2000 pmoles phosphate.
Reactions with the phospho-Thr substrate were done in the following phosphatase assay buffer: 25 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 0.25 mg/ml BSA (31) and allowed to take place for 30 min at 37°C before the malachite green substrate was added. Absorbance was measured following a 15 min incubation at room temperature with

Ni-NTA pull-downs
Five g His 6 -tagged bait protein was allowed to bind to 5 g prey protein in a volume of 0.8 ml pull-down buffer (PDB, 25 mM TrisHCl pH 7.4, 150 mM NaCl, 2 mM DTT, 20 mM imidazole, 0.5% CHAPS), to which 40 l of Ni-NTA slurry pre-equilibrated in PDB was added. Ten g of BSA was added to reduce non-specific binding. After 3h of end-over-end rocking at 4°C, the Ni-NTA beads were pelleted by centrifugation (1 000 g, 2 min 4°C) and washed extensively in PDB. Bound proteins were eluted by boiling the beads in 20 l 2x Laemmli buffer supplemented with 5 mM EDTA. Ten l was loaded on gel. Representative gels of pull-downs repeated at least 3 times are shown.   11 Isoform 1 of Thyroid receptor-interacting protein 13 7 Isoform 1 of HEC1/NDC80-interacting centrosome-associated protein 1 7 HAUS8, Isoform 1 of HEC1/NDC80-interacting centrosome-associated protein 1 7 Isoform ATE1-1 of Arginyl-tRNA-protein transferase 1 6 RCL2, reticulocalbin 2 6 26S proteasome non-ATPase regulatory subunit 3 5 weakly similar to Uro-adherence factor A (Fragment) 5 isoform 1 of CDC42 effector protein 1 5 T complex protein 1 subunit 3 4