Taq Hi-Fi. The reverse primer of fragment 1 contained a sequence for future annealing to the beginning of fragment 2 and the forward primer of fragment 2 contained a sequence for future annealing to the end of fragment 1. Both PCR products were purified on DNA-binding columns (Qiagen, Mississauga, Canada). Then, 2.5 ml of each purified fragment were combined with a master mix containing Taq Hi-Fi buffer, MgSO4, dNTPs and Taq Hi-Fi. Five cycles of PCR amplification were completed under these conditions. During the denaturation step of cycle 5, primers were added: a forward primer containing the HindIII restriction site annealing to the beginning of fragment 1, and a reverse primer containing the EcoRI restriction site annealing with the end of fragment 2. Next, 25–30 additional cycles were completed. The fusion PCR product was purified on agarose gel, then digested with HindIII and EcoRI and cloned into vector pcDNA3.1 (+). Cell culture and transfection The HEK293T cell line was used to produce VLP stocks. This cell line was obtained from the American Type Culture Collection (Manassas, VA) and maintained in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, Burlington, Canada) supplemented with 10% heat-inactived foetal bovine serum (Invitrogen, Burlington, Canada), penicillin G (100 U/ml), and streptomycin (100 mg/ml) at 37uC in 5% CO2. For calcium phosphate transfection, the various plasmids were transfected in 40–50% confluent HEK293T cells in T75 cm2 flasks (BD, Oakville, Canada), as described previously [66,67]. Briefly, fifteen micro- grams of plasmid were used to transfect HEK293T cells and at 6 h post-transfection, the medium was discarded and cells washed twice with phosphate-buffered saline (PBS). Finally, 10 ml of fresh medium was added and the cells were incubated for an additional 48 hours. Preparation of cell lysates and VLP supernatants Cell supernatants were collected 48 h post-transfection, centri- fuged at 4uC for 5 min at 300 6g, and filtered through a 0.22 mM pore-size cellulose acetate membrane to remove cellular debris (ThermoFisher Scientific, Ottawa, Canada). For virus release assays, the cell-free supernatants were centrifuged through a 2 ml 20% sucrose cushions in TSE (10 mM Tris hydrochloride [pH 7.5], 100 mM NaCl, 1 mM EDTA) at 4uC for 45 min at 274,000 6 g (SW41 rotor at 40,000 rpm; 2 ml of cushion for 10 ml of supernatant from each T75 cm2 flask) [21]. The pellets were then resuspended in 150 ml of immunoprecipitation buffer (IPB) (20 mM Tris hydrochloride [pH 7.4], 50 mM NaCl, 1% Triton X-100) containing a protease inhibitor cocktail (Thermo- Fisher Scientific, Ottawa, Canada). The samples were aliquoted and stored at 280uC. As for the transfected cells, they were washed once with 10 ml of room temperature PBS, detached with 10 ml of ice-cold PBS, and pelleted with a brief centrifugation at 4uC for 5 min at 300 6 g. The cell pellets were lysed in 1 ml of IPB containing protease inhibitors, followed by a brief vortexing and microcentrifugation at 4uC for 5 min at 13,000 6 g to remove insoluble materials. Cell lysates were aliquoted and stored at 280uC. All cell lysate samples were normalized with BCA protein quantification assays (Thermo Fisher Scientific, Ottawa, Canada). Gradient SDS-PAGE and Coomassie blue staining Normalized quantities of cell lysates or VLP supernatants were mixed with an equal volume of 2X sample buffer (125 mM Tris hydrochloride [pH 6.8], 4% sodium dodecyl sulfate (SDS), 20% glycerol, 3.1% dithiothreitol (DTT), 0.25% bromophenol blue), and boiled for 5 min at 100uC. Samples were left to migrate on a 7.5–20% gradient SDS-polyacrylamide gel electrophoresis HIV-1 Virus-Like Particles Production (PAGE). Thereafter, the gels were incubated for 15 min in Coomassie Brilliant Blue (Bio-Rad laboratories, Mississauga, Canada) (0.4% R250, 10% acetic acid, 30% methanol), and then discoloured in a mixture of 10% acetic acid and 30% methanol. Gels were digitized with a scanner (SnapScan e20, AGFA). Benchmark Standard ladder protein was used (Invitrogen, Burlington, Canada). Immunoprecipitation (IP) and Western blots analyses To precipitate the Gag protein, we used the monoclonal antibody specific for the major core capsid protein p24 purified from the 183-H12-5C hybridoma (AIDS Repository Reagent Program, Germantown, USA). Briefly, 5 mg of 183-H12-5C antibody were added to cell lysates or cell-free supernatants at 4uC for at least 30 min on a rotary shaker (Labquake, Thermo scientific, Ottawa, Canada). Next, 30 ml of protein A/G plus- Agarose (Santa Cruz Biotechnology, Santa Cruz, USA) were added, and agitated at 4uC for 1 h on a rotary shaker. Next, four washes were done with IPB, followed by microcentrifugation at 4uC for 30 sec at 13,200 6 g. Finally, pellets were resuspended with an equal volume of 26 sample buffer (125 mM Tris hydrochloride [pH 6.8], 4% sodium dodecyl sulfate (SDS), 20% glycerol, 3.1% dithiothreitol (DTT), 0.25% bromophenol blue). After boiling for 5 min, the samples were microcentrifuged for 1 min at 13,000 6 g and transferred to a new tube in order to eliminate beads. Samples were left to migrate on gradient SDS- polyacrylamide gel as described above. After SDS-PAGE migration, proteins were transferred onto Immobilon-P PVDF 0.45 mM membranes (Thermo Fisher Scientific, Ottawa, Canada) by standard blotting technique. Proteins were revealed with a biotinylated anti-p24 antibody (183-H12-5C hybridoma) (EZ-link sulfo-NHS-LC-Biotin Thermo Fisher Scientific, Ottawa, Canada). Primary biotinylated antibod- ies were detected with Cy-3-conjugated streptavidin (Jackson ImmunoResearch Lab, Inc) at a working concentration of 0.5 mg/ ml. Fluorescent signal detection was achieved by scanning the membrane with a fluorescent laser scanner Typhoon 9200 (GE Healthcare Canada, Mississauga, Canada). In order to determine the protein’s molecular weight, the standard protein ladder used was the ECL Plex Fluorescent Rainbow markers (GE Healthcare Canada, Mississauga, Canada). HIV-1 Gag proteins immunode- tected on membranes were quantitated with ImageQuant version 5.2 software, and background correction (local average) was applied for all conditions tested. Graphic was normalized with Pr55Gag wild-type proteins (WT) and expressed as a percentage of WT. Electron microscopy HEK293T cells were transfected with the studied VLP constructs as described above. The cells were harvested 48 h post-transfection, fixed in a solution of 0.1 M cacodylate buffer containing 8% sucrose and 2.5% glutaraldehyde, washed in the same buffer overnight, and then post-fixed with 1% osmium tetroxide at 4uC for 90 min. Dehydration with graded ethanol was then performed, and the cell samples were embedded in Eponate resin (Poly/Bed 812; Polysciences, Burlington, Canada). Sections of 60–80 nm were stained with uranyl acetate and lead citrate, and observed under a JEOL 1010 TEM at a magnification of 50,000, Digital images were processed with the Gatan digital micrograph software version 3.10.1. Indirect immunofluorescence assays Prior to transfection, cells (16104) were plated in 24-well plates (BD, Oakville, Canada) containing German Glass coverslips PLoS ONE | www.plosone.org 11 Month 2011 | Volume 6 | Issue 11 | e28314