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 NL4-3 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 ]
The parent WT virus was generated at Bristol-Myers Squibb from a DNA clone of NL4-3 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 ]
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