Reporter HIV-1 particles were produced by co-transfection of HEK 293T cells with pNL4.3-Env 2 -Luc and either an empty expression vector or an expression vector encoding either a parental or a recombinant env. For each individual recombinant variant, prior to their use for transfection, clones were verified by sequencing of the region encoding the recombinant gene as well as the vector-encoded promoter for its expression. Supernatants, containing virus stock, were harvested 48 h post transfection, and filtered trough a 0.45 mM filter. Production of viral particles was tested using an enzyme linked immunoassay for HIV-p24 antigen detection (Perkin Elmer, MA, USA) and 20 ng of p24 were used to infect 10 5 293T CD4 + -CCR5 + cells in 24 wells plates. Forty-eight hours later, cells were washed twice in PBS and lysed in 25 mM Tris phosphate, pH 7.8, 8 mM MgCl2, 1 mM dithiothreitol, 1% triton X-100, and 7% glycerol for 10 min in a shaker at room temperature. The lysates were centrifuged and the supernatant was used to measure luciferase activity using a GloMax 96 Microplate Luminometer (Promega, WI, USA) following the instruction of the luciferase assay kit (Promega, WI, USA). For samples that yielded negative results in the luciferase assay, plasmids from at least three independent bacterial clones were tested.