Production and Characterization of HIV Variants

Recombinant R5 HIV (HIV_AD8) and X4 HIV (HIV_NL4.3) were prepared by transfecting plasmid encoding full-length of each HIV gene (pAD8 [50 (link)] and pNL4.3 [51 (link)]) into HEK293T cells, respectively. A pseudotyped HIV-1 virus (HIVLuc-VSVG) expressing the luciferase gene (HIV_Luc) was created by transfection of pHIV-1_NL4/3-Luciferase-ΔEnv and p-VSV-G into HEK293T cells in 10 cm dishes [44 (link), 52 (link), 53 (link)]. The HIV-1_NL4/3-Env_AD.8-ΔVpr with Vpr-BLAM was produced by transfection of pHIV-1_NL4/3-Env_AD.8-ΔVpr and pVpr-BLAM [54 (link), 55 (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 [44 (link)]. Supernatants were then ultracentrifuged at 100,000 x g for 2 hours at 4°C onto a 20% sucrose in 10 mM HEPES-150 mM NaCl cushion [56 (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) [49 (link), 56 (link)]. Infection titer (50% tissue culture infectious dose, TCID₅₀) of each virus was determined by an endpoint assay [57 (link)].