Recombinant R5 HIV (HIVAD8) and X4 HIV (HIVNL4.3) were prepared by transfecting plasmid encoding full-length of each HIV gene (pAD8 [45 (link)] and pNL4.3 [46 (link)]) into HEK293T cells, respectively. A pseudotyped HIV-1 virus (HIVLuc-VSVG) expressing the luciferase gene (HIVLuc) was created by transfection of pHIV-1NL4/3-Luciferase-ΔEnv and p-VSV-G into HEK293T cells in 10 cm dishes [42 (link), 47 (link), 48 (link)]. The HIV-1NL4/3-EnvAD.8-ΔVpr with Vpr-BLAM was produced by transfection of pHIV-1NL4/3-EnvAD.8-ΔVpr and pVpr-BLAM [49 (link), 50 (link)] (a kind gift from Dr. Warner Greene) into HEK293T cells. All plasmid DNA transfections were conducted using TransIT-293 (Mirus, Houston, TX, USA) and Opti-MEM I medium (Thermo Fisher Scientific) following a method previously reported [42 (link)]. Supernatants were then ultracentrifuged at 100,000 × g for 2 hours at 4°C onto a 20% sucrose in 10 mM HEPES-150 mM NaCl cushion [51 (link)]. Pelleted particles were resuspended in D10 medium and the concentration of HIV p24 was quantitated by using an HIV-1 p24 ELISA Kit (Perkin Elmer, Boston, MA, USA) [44 (link), 51 (link)]. Infection titer (50% tissue culture infectious dose, TCID50) of each virus was determined by an endpoint assay [52 (link)].
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Production and Characterization of HIV Variants
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Corresponding Organization : Frederick National Laboratory for Cancer Research
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Variable analysis
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- Transfection of plasmid encoding full-length of each HIV gene (pAD8 and pNL4.3) into HEK293T cells
- Transfection of pHIV-1NL4/3-Luciferase-ΔEnv and p-VSV-G into HEK293T cells
- Transfection of pHIV-1NL4/3-EnvAD.8-ΔVpr and pVpr-BLAM into HEK293T cells
- Concentration of HIV p24 quantitated using an HIV-1 p24 ELISA Kit
- Infection titer (50% tissue culture infectious dose, TCID50) of each virus determined by an endpoint assay
- HEK293T cells used for transfections
- TransIT-293 and Opti-MEM I medium used for transfections
- Ultracentrifugation at 100,000 × g for 2 hours at 4°C onto a 20% sucrose in 10 mM HEPES-150 mM NaCl cushion to purify virus particles
- Not explicitly mentioned
- Not explicitly mentioned
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