HIV-1 Molecular Clone Transfection Protocol

HeLa cells (ATCC) were maintained in Dulbecco modified Eagle medium supplemented with 10% (vol/vol) fetal bovine serum (FBS). The Jurkat T cell line (ATCC) was maintained in RPMI 1640 supplemented with 10% (vol/vol) FBS. All media were supplemented with 2 mM l-glutamine. Plasmids used were the infectious HIV-1 molecular clone pNL4-3 (63 (link)); two derivatives with point mutations at SP1 residue 3 (SP1-A3V and SP1-A3T) (39 (link)); four derivatives with point mutations in CA at residues 156 (CA-G165E), 157 (CA-P157S), 160 (CA-P160L), and 225 (CA-G225D) (kind gift from Eric Freed, NIH, USA) (4 (link)); and two protease-defective derivatives, pNL4-3/PR⁻ with the active-site mutation PR-D25N, which contains either wild-type (WT) Gag or the SP1-A3V mutation (64 (link)). Plasmid DNA was purified with a plasmid purification maxiprep kit (Qiagen) and adjusted to 1 μg/μl. HeLa cells were transfected by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and Jurkat T cells were transfected by using DEAE-dextran (65 (link)).