Lipofectamine plus kit

MT-2 cells and the proviral DNA clone NL4-3 were obtained from the NIH AIDS Research and Reference Reagent Program. MT-2 cells were propagated at 37°C/5% CO₂ in RPMI 1640 media (Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco), 100 U/mL of penicillin and 100 μg/mL of streptomycin (Gibco), and sub-cultured twice a week. Virus stocks used to initiate selection were generated by transfecting 293T cells (Lipofectamine PLUS kit, Invitrogen) with proviral DNA clones of NL4-3 Gag P373S (hereafter referred to as wild-type) and NL4_-3 Gag P373S with additional defined Gag amino acid substitutions introduced by site-directed mutagenesis. The Gag P373S substitution was included to better represent the subtype B clinical population: S373 is near the SP1 cleavage site and is present in 60% of subtype B isolates [11 ]. Luciferase reporter variants of NL4-3 (RepRluc Gag P373S) contained the Renilla luciferase (Rluc) gene in the nef locus as previously described [15 (link)].

MT-2 cells were obtained from the NIH AIDS Research and Reference Reagent Program; 293T cells were obtained from the ATCC. Cell lines were sub-cultured twice a week in either RPMI 1640 (MT-2) or DMEM (293T) media (Gibco), supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco), and 100 units/mL penicillin with 100 μg/mL streptomycin (Gibco).
The parent WT virus was generated at Bristol-Myers Squibb from a DNA clone of NL_4-3 obtained from the NIH AIDS Research and Reference Reagent Program[44 ] and contains the Renilla luciferase marker in place of viral nef, and the substitution of Gag P373 for serine, the most common B subtype variation at that position among B subtype viruses (NLRepRlucP373S). NLRepRlucP373S (WT) was modified to contain changes in Gag (for example, V362I, V370A, A364V, ΔV370,[40 (link)] the latter three of which encode high level resistance to BVM.[33 (link), 40 (link), 45 (link)] The recombinant viral DNA was then used to generate virus stocks by transfecting 293T cells (Lipofectamine PLUS kit, Invitrogen). Titers of all stocks were determined in MT-2 cells, using luciferase as the endpoint (Dual-Luciferase Reporter Assay System, Promega).[40 (link), 46 ] The TCID50/ml (tissue culture infectious dose) was calculated by the method of Spearman-Karber.[47 ]

Opossum kidney cells (OKP cells) were maintained in DMEM (Gibco 42430) supplemented with 10% fetal bovine serum (Eurobio, Les Ulis, France), penicillin (100 U/mL), and streptomycin (100 U/mL) at 37 °C in a humidified atmosphere containing 5% CO₂. Human embryonic kidney (HEK) 293 cells were grown in DMEM media complemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For plasmid DNA transfection, cells were grown to 60–70% confluence before being transiently transfected for 5 h using Lipofectamine plus kit according to manufacturer’s instructions (Invitrogen, Paris, France). For protein degradation experiments, cells were treated with MG132 (2 μM) or chloroquine (100 μM) 6 h prior to cell lysis, as previously described [53 (link),61 (link)]. Kifunensine (Sigma k1140, Saint-Quentin-Fallavier, France) was used at 25 µM 6 h before cell lysis.