Plvx zsgreen

HIV-1 NL4-3, NLENY1, NLYUV3, YU-2, lentiviral pLVX-ZsGreen (Clontech), pLVX-Tat, and pLVX-Rev expression vectors were packaged in 293T cells with native HIV-1 envelope or pseudotyped with VSV-G envelope. Viral packaging was done by using 10–17 μg of each lentiviral construct and 3–4 μg of VSV-G plasmid DNA using Lipofectamine plus or Lipofecamine-2000 (Invitrogen) as described earlier (Mehla et al., 2010 (link); Vijaykumar et al., 2008 (link); Zhang et al., 2010 (link)). Lentiviral vectors were packaged using 12 μg pLVX vector, 8 μg gag-pol, 4 μg VSV-G, 3 μg Tat, 4 μg Rev, and 3 μg Vpr plasmids to achieve a workable titer. After 16 h of transfection, cells in 100 mm dishes were washed twice with fresh medium and incubated in RPMI 1640 containing 10% FBS. The culture supernatants were harvested 72–80 h after transfection, centrifuged at 1200 rpm for 15 min, and filtered through a 0.20 μm filter. The filtrate after addition of MgCl2 (4 mM) was digested with 10–50 units of RNase-free DNase (Invitrogen) per 1 μg of plasmid DNA for 30 min at 37°C, aliquoted and stored at −80°C. The viral stocks were quantified for p24 antigen by ELISA (ZeptoMetrix) or titrated for lentiviral particles on reporter cells. A viral inoculum of 50–1000 ng/mL was used for infection of Jurkat, primary macrophages, HFA, and SVGA.