Expression of HIV-1 pol in insect cells
The Pol coding region from pNL4-3 (nucleotides 2091 to 5093) was introduced between the EcoRI and XhoI sites of pFastBac1 (Life Technologies). A His6 tag coding sequence has been appended at the C-terminus of Pol. Recombinant bacmids and baculoviruses were obtained as previously described [18]. Baculoviruses were used to infect 2 l of High Five cells (Life Technologies) grown in suspension in Express Five SFM medium (Life Technologies). After 56 h of culture at 27 °C, cells were harvested by centrifugation, washed with ice-cold buffer 150/10 (20 mM K-phosphate pH 7.5, 150 mM NaCl, 10 mM imidazole, 5% glycerol, 5 mM 2-mercaptoethanol), and the cell pellet was stored at − 80 °C. Cells were lysed at 37 °C after addition of 30 ml of buffer 150/10 containing 1% Triton X-100, in the presence of 1 mM Pefabloc, 10 mM benzamidine and 10 mM PMSF. After addition of 60 ml of buffer 500/50 (20 mM K-phosphate pH 7.5, 500 mM NaCl, 50 mM imidazole, 5% glycerol, 5 mM 2-mercaptoethanol), extract was clarified by centrifugation at 70,000 g for 30 min at 4 °C and incubated 1 h at 4 °C with 0.5 ml of Ni-NTA Superflow matrix (Qiagen). Beads were extensively washed with buffer 500/50, and elution was performed by adding 5 × 1 ml of buffer 500/400 (20 mM K-phosphate pH 7.5, 500 mM NaCl, 400 mM imidazole, 5% glycerol, 5 mM 2-mercaptoethanol). Eluate was concentrated by ultrafiltration (Vivaspin 6, 10 kDa) to a volume of 0.5 ml and applied to an TSK G4000 SW column (300 × 7.5 mm) equilibrated in 20 mM Hepes pH 6.8, 250 mM NaCl, 2% glycerol and 2 mM DTT. Fractions containing Pol were concentrated by ultrafiltration (Vivaspin 6, 10 kDa), and stored at − 80 °C. Protein concentration was determined by using a calculated absorption coefficient of 1.786 A280 units mg− 1 cm2.
Expression of HIV-1 integrase in insect cells
The IN coding region from pNL4-3 (nucleotides 4230 to 5093) was introduced between the EcoRI and XhoI sites of pFastBac1 (Life Technologies). A His6 tag coding sequence has been appended at the C-terminus of integrase. Recombinant bacmids and baculoviruses were obtained as previously described [18]. Baculoviruses were used to infect 8 l of High Five cells (Life Technologies) grown in suspension in Express Five SFM medium (Life Technologies). After 68 h of culture at 27 °C, cells were harvested by centrifugation, washed with ice-cold buffer 150/10, and the cell pellet was stored at − 80 °C. Cells were lysed at 37 °C after addition of 120 ml of buffer 150/10 containing 1% Triton X-100, in the presence of 1 mM Pefabloc, 10 mM benzamidine and 10 mM PMSF. After addition of 240 ml of buffer 500/50, extract was clarified by centrifugation at 70,000 g for 30 min at 4 °C and incubated 1 h at 4 °C with 1 ml of Ni-NTA Superflow matrix (Qiagen). Beads were extensively washed with buffer 500/50, and elution was performed by adding 5 × 1 ml of buffer 500/400. Eluted proteins were dialyzed against buffer ASU (20 mM Tris-HCl pH 7.0, 150 mM NaCl, 1 M urea, 10% glycerol, 1 mM EDTA, 10 mM 2-mercaptoethanol) and applied to a Mono S HR 5/5 column (GE Healthcare) equilibrated in the same buffer. Proteins were eluted by a linear gradient (40 column vol.) of NaCl from 150 to 450 mM. Fractions containing integrase were concentrated by ultrafiltration (Vivaspin 6, 10 kDa), dialyzed against storage buffer (20 mM K-phosphate pH 7.5, 1 M NaCl, 2 mM DTT), and stored at − 80 °C. Protein concentration was determined by using a calculated absorption coefficient of 1.529 A280 units mg− 1 cm2.
Expression of HIV-1 transframe in E. coli
The transframe (TF) coding region from pNL4-3 (nucleotides 2091 to 2252) was codon-optimized for expression in E. coli and introduced between the NcoI and XhoI sites of pET28b. A His6 tag coding sequence has been appended at the C-terminus of TF. Expression of TF was conducted in E. coli BL21(DE3) (Invitrogen) grown at 37 °C in 8 l of LB medium supplemented with kanamycin (50 μg/ml). When the culture reached an A600 = 0.3, temperature was adjusted to 20 °C and expression was induced by addition of 1 mM IPTG for 4 h when A600 was equal to 0.5. Cells were washed twice with ice-cold buffer 150/10, resuspended in 60 ml of the same buffer containing 1 mM Pefabloc, 10 mM benzamidine and 10 mM PMSF, and lysed by sonication. All subsequent steps were conducted at 4 °C. Extract was clarified by centrifugation at 70,000 g for 30 min and incubated 1 h at 4 °C with 1 ml of Ni-NTA Superflow matrix (Qiagen). Beads were extensively washed with buffer 500/50, and elution was performed by adding 5 × 1 ml of buffer 500/400. Eluted proteins were dialyzed against buffer AS (20 mM Tris-HCl pH 7.5, 10 mM KCl, 10% glycerol, 10 mM 2-mercaptoethanol), and applied to a Mono S HR 5/5 column equilibrated in the same buffer. Proteins were eluted by a linear gradient (40 column vol.) of KCl from 10 to 300 mM. Fractions containing TF were concentrated by ultrafiltration (Amicon Ultra-4, 3 kDa), dialyzed against PBS (136 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.47 mM KH2PO4), and stored at − 80 °C. Protein concentration was determined by using a calculated absorption coefficient of 0.773 A280 units mg− 1 cm2.
The apparent native molecular mass of TF was determined by gel filtration on a Yarra 3u SEC-2000 column (300 × 4.6 mm) (Phenomenex) equilibrated in 200 mM K-phosphate (pH 6.8), 5 mM 2-mercaeptoethanol, and developed at room temperature at a flow rate of 0.05 ml/min. The calibration curve was established by using cytochrome C, ovalbumin, and bovine serum albumin as marker proteins. For a particular protein, its elution was described in term of the corresponding Kav value. Kav = (Ve-V0)/(Vt-V0), where Ve is the elution volume of the particular molecule, V0 the void volume of the column, and Vt the total bed volume. V0 and Vt were determined with dextran blue (> 5 MDa) and vitamin B12 (1.35 kDa), respectively.
Expression of TF-Sx-IN surrogates of pol in E. coli.
The TF and IN sequences from pNL4-3 were introduced into pET28b with a BamHI site between the TF and IN coding regions. The BamHI site encodes for a 2 residue spacer, S2 (Gly-Ser), leading to the expression of the TF-S2-IN-H6 fusion protein. An oligonucleotide duplex (5’-GATCTGGGGGTGGCG and 5’-GATCCGCCACCCCCA, encoding a GGGGS peptide) was recursively introduced into the BamHI site to give pET28b/TF-S7-IN-H6 (one insert), pET28b/TF-S12-IN-H6 (two inserts), pET28b/TF-S17-IN-H6 (three inserts) and pET28b/TF-S22-IN-H6 (four inserts).
Expression of the TF-Sx-IN fusion proteins was conducted in E. coli BL21(DE3) (Invitrogen) grown at 37 °C in 6 l of LB medium supplemented with kanamycin (50 μg/ml). When the culture reached an A600 = 0.5, expression was induced by addition of 1 mM IPTG for 4 h. Cells were washed twice with ice-cold buffer 150/10, resuspended in the same buffer (1 ml per g of cell pellet) containing 1 mM Pefabloc, 10 mM benzamidine and 10 mM PMSF, and lysed in an Eaton Press after freezing in dry ice. All subsequent steps were conducted at 4 °C. After addition of 2 vol. of buffer 150/10, extracts were clarified by sonication and by centrifugation at 70,000×g for 30 min. After incubation 1 h at 4 °C with 1 ml of Ni-NTA Superflow matrix (Qiagen), beads were extensively washed with buffer 500/50, and elution was performed by adding 5 × 1 ml of buffer 500/400. Eluted proteins were dialyzed against buffer 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 M urea, 10% glycerol, 1 mM EDTA, 10 mM 2-mercaptoethanol, and applied to a Mono S HR 5/5 column (GE Healthcare) equilibrated in the same buffer. Proteins were eluted by a linear gradient (40 column vol.) of NaCl from 100 to 400 mM. Fractions containing the TF-Sx-IN fusion proteins were adjusted to 0.02% Triton X-100, concentrated by ultrafiltration (Vivaspin 6, 10 kDa), dialyzed against storage buffer (20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5% glycerol, 10 mM 2-mercaptoethanol, 0.02% Triton X-100), and stored at − 80 °C. Protein concentration was determined by using the BioRad Protein Assay.
Antibodies and western blot analysis
Rabbit anti-TF antibodies were generated against a synthetic peptide (KAREFSSEQTRANSPTRRE) corresponding to residues 10-28 of HIV-1 transframe protein (Life Technologies). Western blot analyses were conducted with goat anti-rabbit secondary antibodies conjugated with peroxidase (Chemicon) and the SuperSignal West Pico chemiluminescent substrates (Thermo Scientific).
HTRF assay
Homogeneous time-resolved fluorescence (HTRF) assays were performed in black, half-area, 96-well microplates. Mitochondrial LysRS (mLysRS) or a derivative with a C-terminal deletion of 22 aminoacid residues (mLysRS∆C) were expressed in E. coli with a C-terminal HA-tag (YPYDVPDYA), and purified as described [12]. mLysRS-HA (1.5 nM, dimer concentration) was incubated with various concentrations of Pol-H6 (0.02 to 10 nM, dimer concentration), IN-H6 (0.5 to 125 nM, dimer concentration) or TF-H6 (1 to 1000 nM, monomer concentration) in 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM 2-mercaptoethanol and BSA at 1 mg/ml, for 1 h on ice. For the determination of the binding affinities of the TF-Sx-IN surrogates, the buffer was supplemented with 0.02% Triton X-100. Antibodies (Cisbio) directed to the His-tag, conjugated with Eu3+ cryptate, and to the HA-tag, conjugated with XL665, were added and incubation was continued for 30 min. After addition of 50 mM KF, fluorescence of Eu3+ cryptate and of XL665 was recorded at 620 nm (I620) and 665 nm (I665), respectively, after excitation of Eu3+ cryptate at 317 nm, in an Infinite M1000 PRO microplate reader (TECAN). Results are expressed as the ratio of I665/I620.
Circular dichroism spectroscopy
CD spectra were recorded at 20 °C or 90 °C with a path length of 1 mm in a Jasco J-810 apparatus equipped with a Peltier temperature controller. Each spectrum is a mean of 10 scans. Protein was dialyzed against 25 mM K-phosphate (pH 7.5) and its final concentration was 12 μM. Spectra were analyzed using the Dichroweb software [21].
Fluorescence polarization assay
Fluorescence polarization was measured in an Infinite M1000 Pro reader (TECAN) after incubation for 1 h on ice in PBS buffer containing 0.02% Triton X-100. Mitochondrial LysRS (mLysRS) was expressed in insect cells and purified as described [12]. tRNA3Lys-Cy3 was synthesized in vitro with Cyanine3 attached at the 5′-extremity (eurofins). Renaturation of tRNA was performed in 5 mM MgCl2 after heating at 90 °C for 2 min, and slow cooling at 25 °C. The efficiency of renaturation was measured in the tRNA aminoacylation reaction (420 pmol/A260).