Jurkat cells (CD4+ T cells) were maintained in RPMI 1640 medium (Wisent) supplemented with 10% (v/v) FBS (Wisent) and HEK 293T cells (human embryonic kidney cells transformed with adenovirus and simian virus 40 large-T) were maintained in DMEM (Gibco) supplemented with 10% (v/v) FBS. Transfections were performed with polyethylenimine (PEI) (Polysciences, Inc.) in six-well plates containing Jurkat cells (1.2 Â 10 6 ), 293T cells (4.0 Â 10 5 ) or 293T stable transfectants (6.0 Â 10 5 cells) expressing a dual-luciferase HIV reporter (see subsequently). PEI was added drop-wise to serum-free medium and incubated 10 min at room temperature. In parallel, serum-free medium was added to DNA. The diluted PEI was added to the DNA solution (PEI to DNA ratio of 2:1) and incubated at least 15 min at room temperature. An empty plasmid, pcDNA3.1Hygro+, was added, when required, to maintain an equivalent DNA input.